A rapid and efficient platelet purification protocol for platelet gene expression studies Stefan Amisten Address: Oxford Centre for Diabetes, Endocrinology and Metabolism, Oxford University, The Churchill Hospital, Headington, Oxford, OX3 7LJ, U.K. Email: stefan.amisten@gmail.com

Abstract Isolation of pure platelet samples from whole blood is crucial for the study of platelet gene expression. The main obstacles to overcome in order to successfully isolate platelets from whole blood include: 1) platelet activation; 2) leukocyte and red blood cell contamination and 3) time-dependent platelet mRNA degradation. This chapter describes a rapid and highly efficient method for isolating human circulating platelets from small volumes of whole blood based on efficient inhibition of platelet activation and leukocyte removal by filtration followed by magnetic bead-depletion of residual contaminating leukocytes and red blood cells. Also described are methods for RNA extraction, cDNA synthesis and platelet gene expression studies using both quantitative real-time PCR and microarray. Key Words: platelet purification, platelet activation, leukocyte-depletion, gene expression, real-time PCR, qPCR, microarray, RNA extraction. 1. Introduction Microarray and real-time PCR have both been used to study the platelet transcriptome(1-5). However, successful platelet gene expression profiling is often hampered by the contamination of platelet preparations by other cell types such as leukocytes. Platelets are anuclear and, as a consequence, all non-mitochondrial platelet mRNA is derived from their precursor cell, the megakaryocyte. As a result, the amount of mRNA a platelet contains is dependent on the amount of mRNA inherited when it budded off from the megakaryocyte as well as the rate of platelet RNA degradation. Therefore, when studying platelet gene expression, it is advantageous to use a fast platelet purification method in order to minimize further degradation of mRNAs during the purification process. Also, it is important to minimize the contamination of the platelet preparations by other cell types such as red blood cells and leukocytes. This is particularly important for leukocytes, which have been reported to contain as much as 10,000 times more RNA than platelets(6). This chapter describes a fast, flexible and low cost method for the generation of a highly purified population of human platelets from less than 100 mL of whole blood(1) (see Note 1). The protocol combines efficient inhibition of platelet activation with leukocyte removal using filters developed for the preparation of platelets for clinical use (7) and antibody-mediated magnetic depletion of residual leukocytes and erythrocytes. The purification can be performed in less than two hours and without the need for expensive or bulky specialized equipment. The chapter also describes the use of real-time PCR to assess the expression of two platelet P2Y receptors (P2RY1 and P2RY12) and the level of residual contaminating leukocyte-specific messages (CD45). 2. Materials 2.1 Platelet isolation

1. Dynabeads® Pan Mouse IgG (Invitrogen) 2. Mouse anti-human CD235a (red blood cell surface marker) (BD Inc, NJ, USA) 3. Mouse anti-human CD45 (leukocyte surface marker) (BD Inc, NJ, USA) 4. Dynabead-compatible magnet (DynaMag™-2 or DynaMag™-15) (Invitrogen) PBS, pH 7.4

(Invitrogen) (see Note 2) 5. Dynabead wash buffer: PBS with 0.1% (w/v) BSA, pH 7.4 6. Sterile anticoagulant: citrate-dextrose solution (ACD) (Sigma Aldrich) 7. Prostaglandin E1 (PGE1) (Sigma Aldrich): 1 mM stock in ethanol 8. Acetylsalicylic acid (Sigma Aldrich): 30 mM stock in ethanol 9. EDTA, 0.5M (Sigma Aldrich) 10. 50 and 15 mL centrifuge tubes and 1.5 and 2 mL Eppendorf tubes (see Note 2)

11. Intravenous venflon cannula 18 gauge with port and flexible tubing (Becton Dickinson Infusion Therapy AB, Sweden)

12. Rotating and tilting test tube carousel 13. AutoStop™ BC high efficiency filter for leukocyte removal (Pall Inc, Cat no ATSBC1E, see

http://www.pall.com/medical_48187.asp) 14. TRIzol (Invitrogen)

2.2 RNA purification and cDNA generation

1. RNase free filter tips: 10 μL, 100 or 200 μL and 1000 μL 2. RNase free tubes 1.5 mL and 0.6 mL (Axygen MCT-060-A and MCT-150-C) 3. Isopropanol, molecular biology grade (SigmaAldrich) 4. Ethanol, molecular biology grade (SigmaAldrich) 5. Water, molecular biology grade (ie. RNase-free) (SigmaAldrich) 6. Chloroform, molecular biology grade (SigmaAldrich) 7. UltraPure™ Glycogen (Invitrogen, Cat. No. 10814-010) 8. PCR tubes, RNase free 9. TaqMan reverse transcription kit (Applied Biosystems) 10. RNeasy MinElute Cleanup Kit (Qiagen) 11. Centrifuge with cooling for spinning at 12000 x g 12. NanoDrop (Thermo Scientific) or equivalent spectrophotometer for assaying nucleotide

content in small sample volumes 13. Optional - 2100 Bioanalyzer (Agilent Technologies)

2.3 Quantitative real-time PCR

1. QuantiFast SYBR Green PCR Kit (Qiagen) 2. QuantiTect Primer Assays (Qiagen):

a. P2RY1 (QT00199983) – platelet ADP receptor used to demonstrate the qPCR protocol b. P2RY12 (QT01770111) – platelet ADP receptor used to demonstrate the qPCR protocol c. ACTB (QT01680476) – housekeeping gene d. P2RY11 (QT01150282) – GPCR expressed on leukocytes but not on platelets e. CD45 (PTPRC) (QT01869931) – abundant leukocyte protein, not present on platelets

3. PCR tubes 4. Filter tips, PCR grade 5. Equipment for agarose gel electrophoresis of 50 – 200 bp qPCR products

3. Methods The platelet purification method has four main phases: blood collection, generation of platelet rich plasma (PRP), PRP filtration and magnetic bead-mediated leukocyte and erythrocyte depletion. Appropriate ethical permission to take human blood should be obtained. To minimize platelet activation during blood collection, a 1.2 mm diameter (18 gauge) intravenous cannula is inserted into a suitable vein, and the blood is collected through the attached tubing via gravity flow into the platelet inhibition cocktail. In the author’s experience, this method is superior to collecting blood using vacutainers, as it reduces mechanical platelet activation caused by the vacuum. Once purified, the platelet samples are dissolved in TRIzol and frozen until RNA extraction. 3.1. Preparation of antibody-conjugated magnetic beads

1. Mix 1 mL Dynabead wash buffer and 250 l Dynabead slurry. 2. Place the tube in the Dynamag magnet for 1 min, remove the liquid and repeat for a total of

two washes. 3. Remove the washed beads from the magnet and resuspend the washed Dynabeads up to the

original bead volume (i.e. 250 μL) using Dynabead wash buffer

4. Add 15 l of anti-CD235a antibody and 15 l anti-CD45 antibody to the beads and mix. 5. Incubate the bead mix for at least 30 min with gentle tilting and rotation. 6. Place the tube in the magnet for 1 min and remove the supernatant. Resuspend the beads up

to the original bead volume (i.e. 250 μL) using Dynabead wash buffer and store the antibody-conjugated magnetic beads at 4oC.

3.2. Preparation of platelet inhibition cocktail The volumes stated below apply to 100 mL whole blood collected into twelve 15 mL tubes.

1. Mix 18 mL ACD, 12 l of 1 mM PGE1, 120 l of 30 mM Acetylsalicylic acid and 480 l of 0.5M EDTA

2. Add 1.5 mL of the cocktail into each of the twelve 15 mL tubes 3.3. Collection of blood and purification of platelets

1. Weigh an empty 15 mL tube with cap and note down the tube weight. 2. Implant the intravenous cannula (1.2 mm diameter) and attach the flexible tubing. Collect 8.5

mL of blood by self propagated flow through the flexible tubing into each tube containing 1.5 mL platelet inhibition cocktail. When the total volume reaches 10 mL, replace the cap and invert the tube gently to enable thorough mixing of the blood with the platelet inhibition cocktail. To fill 12 tubes you will need 102 mL of whole blood (see Notes 1, 3-4).

3. Place tubes in a centrifuge and spin at room temperature (200 x g, 20 min) 4. Carefully transfer the top 85% of the upper layer, the platelet rich plasma (PRP), to new 15

mL tubes. 5. Spin at room temperature (200 x g, 10 min) to further remove leukocytes and red blood cells. 6. Carefully transfer the top 85% of the PRP to a 50 mL tube. 7. With a sterile pair of scissors or a surgical blade, cut off excess tubing from the Pall Leukocyte

removal filter pack and mount it on a rack. 8. Place the drainage tubing in a 50 mL collection tube and manually inject the PRP into the

filter at a flow rate of 15 mL per minute. Collect the filtrate in a 50 mL tube. 9. Add all of the antibody-conjugated magnetic beads to the tube containing the filtered PRP

and place the tube in a tilting and rotating carousel at room temperature for 45 minutes. 10. Transfer the PRP/bead mix into 2 mL tubes (see Note 2) and leave the tubes in the

DynaMag™-2 for 2 minutes. The DynaMag™-2 can accommodate 16 tubes at a time, so steps 10-13 will have to be repeated if more than 32 mL of PRP is used.

11. With the tubes still held in the magnetic rack, transfer the supernatants into new magnet compatible tubes without disturbing the magnetic beads that are held by the magnetic field.

12. Put the tubes containing the depleted PRP in the magnet. 13. After 2 min, without disturbing the beads captured by the magnetic field, transfer the

supernatant into new 15 mL tubes and harvest the platelets by centrifugation at 800 x g, 10 minutes at room temperature

14. Discard the supernatants. Weigh the 15 mL tubes containing the platelet pellets and calculate the weight of each platelet pellet by subtracting the empty tube weight from step 1 above. Dissolve the platelet pellets using 1 mL TRIzol per 100 mg platelets (use a minimum of 1 mL TRIzol for platelet samples weighing less than 100 mg) and leave for 5 minutes at room temperature (see Note 5).

15. Transfer the dissolved platelets into 1.5 mL tubes (1 mL per tube) and store the samples at -

80 C until RNA purification. RNA samples in TRIzol are stable for up to 1 month at -80 C or below.

3.4. Extraction of platelet total RNA The following protocol for isolation of platelet total RNA is based on a modified version of the TRIzol method(1). An advantage of the TRIzol method over column-based RNA purification methods is that besides high quality RNA, platelet protein may also be extracted from the same platelet TRIzol

sample (see Notes 5-9). The RNA isolated using TRIzol is then further purified and concentrated using an RNeasy MinElute Cleanup Kit (Qiagen). The RNeasy MinElute Cleanup Kit columns remove contaminating residual salts and phenol as well as RNAs shorter than 200 base pairs, such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise 15–20% of the total RNA, resulting in an enrichment of mRNAs(8).

1. Cool isopropanol to -20 C.

2. Cool centrifuge to 4 C.

3. Place platelet samples dissolved in TRIzol in a fume hood, defrost and leave for 5 min at room temperature.

4. Add 200 l chloroform to each tube containing 1 mL TRIzol (see Notes 5 and 10).

5. Manually shake the tubes 15-30 times and leave for 2-3 min at room temperature

6. Spin at 12000 x g for 15 min at 4 C

7. Add 10 μg ultra-pure glycogen (0.5 μl of 20 μg/μL stock solution) to new RNase free tubes (see Note 11)

8. Transfer the aqueous phase of each tube containing TRIzol/chloroform mix to tubes containing glycogen using a 1000 μL filter tip. Leave about 3-4 mm of the aqueous phase above the interphase (figure 1) to minimize carryover of contaminating DNA.

9. Add 500 l of cooled (-20 C) isopropanol to each tube of aqueous phase/glycogen mix

10. Invert to mix and leave over night at -20 C or at -80C for 1 hour

11. Spin the samples at 12000 x g for 15 min at 4 C

12. Remove all the liquid and add 1 mL of 75% ethanol (freshly made up with molecular biology grade water). RNA pellets from the same donor that were split into separate 1 mL aliquots in TRIzol (steps 14 and 15, section 3.3) may be combined by pooling the RNA pellets into one tube in a total volume of 1 mL 75% ethanol. RNA samples in 75% ethanol can be stored at -

20 C for at least one year.

13. Spin at 7500 x g for 10 min at 4 C

14. Remove all the liquid. It is important to remove ALL the liquid, otherwise it will take a very long time to dry the RNA pellet.

15. Dry the pellets by placing the tubes in a heating block at 35-37 C for 1-3 min with the lids open.

16. When the RNA pellet appears dry, dissolve the RNA in 101 μL water, vortex and keep on ice.

17. Transfer 1 μL RNA into an RNase-free 1.5 mL tube containing 3 μL RNAse free water (makes 4 μL of 1 in 4 diluted RNA). Freeze the remaining 100 μL RNA at -80C until RNeasy MinElute Cleanup column purification.

18. Use 1 μL of the 1 in 4 diluted RNA to measure the total RNA concentration on a NanoDrop or equivalent spectrophotometer.

19. Calculate the total amount of RNA present in each sample and purify each RNA sample using the RNeasy MinElute Cleanup kit according to the manufacturer’s instructions. Do not load more than 45 μg RNA dissolved in 100 μL RNase free water on each spin column(8).

20. The total elution volume from the RNeasy MinElute column is 12 μL. Transfer 0.5 μL of the RNeasy MinElute kit purified RNA into a new RNase free tube containing 1.5 μL molecular grade water (makes 2 μL of 1 in 4 diluted purified RNA). Store the remaining 11.5 μL of RNA at -20oC to -80oC until cDNA synthesis.

21. Use 1 μL of the 1 in 4 diluted RNA to measure the RNA concentration on a NanoDrop or equivalent. Calculate the concentration of the undiluted purified RNA sample. The purified RNA can be used for both cDNA synthesis and microarray hybridization. However, it is recommended that the RNA integrity of all samples intended for microarray hybridizations are verified using a 2100 Bioanalyzer (Agilent Technologies).

3.5. Generation of platelet cDNA Reverse transcription of RNA into cDNA can be achieved using a variety of kits. In our hands, the TaqMan reverse transcription kit (Applied Biosystems) has worked very well, and it will be used as an example below. The TaqMan reverse transcription kit is designed to transcribe 60-2000 ng of total RNA into cDNA. If larger amounts of RNA are needed, it is recommended that several cDNA reactions not exceeding 2000 ng are performed, followed by pooling of all the cDNA reactions into one.

1. Program the thermal cycler for reverse transcription PCR (table 1). 2. Prepare a reverse transcription master mix (table 2) and distribute to RNase and DNase free

PCR tubes (see Note 12). Add 60-2000 ng RNA to each tube, vortex and spin down. Always keep all samples and reagents on ice. The total reaction volume can be scaled up or down according to the amount of cDNA needed.

3. Place PCR tubes containing reverse transcription mix and RNA in the thermal cycler. Ensure that the PCR tube caps are tightly closed.

4. Once the thermal cycling is completed, the cDNA can be stored short-term at 4 C or long

term at -20 C 3.6 Relative quantitative real-time PCR (qPCR) of platelet genes To illustrate the use of quantitative real-time PCR (qPCR) the following protocol describes the quantification of the platelet ADP receptor genes P2RY1 and P2RY12 relative to the house keeping

gene beta-actin (ACTB). The approach uses a Rotor-Gene 6000 real-time PCR cycler, the QuantiFast SYBR Green PCR Kit and QuantiTect primer assays (all from Qiagen) (see Note 13-14). To evaluate the degree of residual leukocyte contamination, two leukocyte specific genes may also be quantified: PTPRC, which encodes the leukocyte surface protein CD45, and P2RY11, which encodes a G-protein coupled receptor that is expressed on leukocytes but not on platelets. The degree of leukocyte depletion in the purified platelet samples can easily be measured by qPCR quantification of CD45 in cDNA from whole blood or PRP (from step 6 in section 3.3) compared to purified platelets from step 13 in section 3.3).

1. Program the Rotor-Gene 6000 thermal cycler (table 3 and Note 15). 2. Prepare a master mix for pilot qPCR quantification (table 4 and Note 16). Keep all samples on

ice and avoid exposing your mastermix to light, as the SYBR Green in the mastermix is light sensitive. Increased qPCR sensitivity and reduced reaction cost can be achieved by reducing the total reaction volume from 25 μL (i.e. the volume recommended by Qiagen) to 10 or 5 μL without reducing the total amount of cDNA template added to each qPCR reaction (see Note 16). For the pilot qPCR it is recommended to pool cDNA from each biological replicate and to dilute the pooled cDNA at least 1 in 4, as this approach will use up less cDNA from each biological replicate. Example: Platelet cDNA has been generated from 3 different platelet donors. For the initial pilot qPCR of the genes P2RY1, P2RY12 and ACTB, 1 μL of 1 in 4 diluted pooled cDNA will be used for each 10 μL reaction. It is very hard to predict the optimal cDNA dilution for each gene, since the expression of two different genes can vary by several orders of magnitude. Therefore, by running a pilot qPCR using a standard dilution it is possible to determine the optimal cDNA dilution factor required for the quantification of each gene of interest. In order to include cDNA from all the three platelet samples, 0.5 μL cDNA is pooled from each platelet sample, giving a total volume of 1.5 μL pooled platelet cDNA. For a 1 in 4 dilution of the cDNA, 4.5 μl water is added, resulting in 6 μL of 1 in 4 diluted pooled platelet cDNA. 3.5 μL of this cDNA is used for the pilot qPCR (see table 4), and the remaining is frozen for future use.

3. Close the lids tightly, place the samples in the thermal cycler and run the program set up in step 1.

4. Perform a melt curve analysis for each primer assay to verify that only one amplification product has been produced in each qPCR sample. Please see the product documentation included with the QuantiFast SYBR Green PCR Kit and your qPCR thermal cycler for specific instructions on how to perform a melt curve analysis. If a gene of interest is not expressed in your sample, or if it is present at only very low levels, the QuantiTect primer assay may generate a non-specific qPCR product (figure 2b). These non-specific amplifications will be detected by the melt curve analysis and by agarose gel electrophoresis of the qPCR samples. Due to the small size of the qPCR products (50-200 bp), a 2% agarose gel is recommended for clear resolution of the individual bands. Only primer assays that generate a single melt curve peak (Fig. 2a) and a single qPCR product corresponding to the size given in the QuantiTect primer assay product literature should be used.

5. Calculate the Ct (Cycle threshold) values for all the samples in the qPCR run. Please see the product documentation included with the QuantiFast SYBR Green PCR Kit and your qPCR thermal cycler for specific instructions on how to calculate the Ct values. In our hands, when using the Rotor-Gene 6000 and the RotorGene version 6.0 software, threshold values between 0.02 and 0.05 often give satisfactory Ct values.

6. Determine the optimal dilution of your cDNA samples for the quantification of your genes of interest. By diluting the cDNA, it is possible to save platelet cDNA without compromising the quality of the qPCR quantifications (see Note 17). Example: The pilot quantification using pooled cDNA diluted 1 in 4 (see steps 2-5 above) returned the following Ct values: P2RY1: 25.91; P2RY12: 23.26; ACTB: 18.79. Since all Ct values are between 17 and 30 cycles, no further dilution of the cDNA is necessary, and a 1 in 4 dilution of the cDNA is sufficient for the quantification of all three genes. However, if there is a need to conserve cDNA, the cDNA used to quantify ACTB can be diluted up to 16 times more and P2RY12 twice more without compromising the qPCR quality.

7. An ideal primer pair has an amplification efficiency (E) of 2 (i.e. each PCR cycle results in a

doubling of the number of template DNA molecules). However, sometimes the E value deviates slightly from the theoretical value. In order to determine the actual amplification efficiency for each primer assay, each gene needs to be quantified using serially diluted cDNA. The Ct values from the dilution series is then used to calculate the amplification efficiency for each gene: E = 10(-1/k) where k is the slope of the linear function of the Ct values plotted against the log transformed cDNA dilution factors. Most functional primer assays will generate primer efficiencies (E) of 1.9-2.1 (figure 3a), whereas non-functional primer assays will not generate straight Ct vs. log dilution factor curves (figure 3b) or primer efficiencies much greater or smaller than 2. To determine the E value for a primer assay, prepare serially diluted cDNA (see Table 7) and quantify each gene in triplicate as described in steps 2-6 above. For each gene, prepare a master mix for 17 x 10 μL qPCR reactions (see table 5). Example: The primer efficiency (E) for the primer assays P2RY1, P2RY12 and ACTB needs to be determined experimentally using serially diluted platelet cDNA. The required volumes for the evaluation of three primer assays in triplicate can be calculated as shown in table 6 and table 7.

The average Ct values for each dilution step are plotted in a graph against the log values of the dilution factors and the equation for the linear trendline is calculated (table 8 and figure 3a). Finally, the amplification efficiency (E) for each primer assay is calculated according to the formula E=10(-1/k). Since k (i.e. the slope of the trendline) for the ACTB primer assay dilution curve is -3.3917, the amplification efficiency (E) for the ACTB primer assay is: E = 10(-1/-3.3917) = 1.97 Using the same method, the E values for the P2RY1 and P2RY12 primer assays are found to be 2.01 and 1.98.

8. Make a new mastermix (table 9) and quantify all genes of interest. It is recommended that at least three biological replicates are used and that each gene is quantified at least in duplicate reactions. In order to detect subtle changes in gene expression between samples and controls, it is recommended to use at least eight biological replicates in each group.

Example: A total of 18 reactions are run for the quantification in duplicates of P2RY1, P2RY12 and ACTB using platelet cDNA from three individuals, resulting in the following Ct values for platelet cDNA samples 1-3: P2RY1: 25.98, 25.32, 25.79; P2RY12: 22.7, 22.72, 22.92; ACTB: 18.58, 18.43, 18.6.

9. Calculate the expression of your genes of interest relative to your housekeeping gene using the formula: Relative expression (R) = (Egene of interest

-Ct gene of interest) / (Ehousekeeping gene-Ct housekeeping gene)

Example: The expression of P2RY1and P2RY12 relative to ACTB in platelet cDNA sample 1 is:

RP2RY1= (2.01-25.98) / (1.97-18.58) = 0.0039 RP2RY12= (1.98-22.7) / (1.97-18.58) = 0.054

3.6 Preparation of RNA samples for platelet microarray analysis There are many different microarray platforms available, and some of the more commonly used ones are Illumina and Affymetrix. All microarray platforms have their pros and cons, and in most cases, the choice of microarray platform is based on cost and which microarrays are available locally. Many academic institutions now have core facilities specializing in microarray hybridizations and data analysis, and it is highly recommended to let your local core facility handle all microarray hybridizations. If insufficient platelet RNA is available, it may also be necessary to either pool RNA from several different platelet preparations(9-12) or to perform a double amplification of the platelet RNA (13). Both methods have their pros and cons, and it is up to the researcher and the local microarray core facility to find an optimal solution suitable for the intended project and the local conditions. In order to validate gene expression results obtained by microarray, it is recommended that 5-10% of the total amount of the RNA intended for microarray hybridization is set aside for cDNA synthesis and qPCR validation of the microarray results.

4. Notes

1. The expected platelet yield may vary between donors. In general, dehydrated donors will give a lower proportion of platelet rich plasma compared to the red blood cell fraction than well hydrated donors. To increase platelet yield, it is therefore recommended that all blood donors are well hydrated prior to blood donation. Various diseases and medications may also affect the platelet count, which will influence the final platelet yield.

2. Depending on the type of magnet that is used (DynaMag™-2 or DynaMag™-15), either 1.5 or 2 mL tubes (DynaMag™-2) or 15 mL tubes (DynaMag™-15) are suitable for magnetic cell depletion. For the sake of consistency, 2 mL tubes are stated throughout this protocol.

3. Self propagated blood flow is preferred in order to minimize platelet activation during the blood collection process.

4. Platelet yield and gene expression profiles may show natural variations, in addition to variations due to different disease states, medication and pregnancy.

5. TRIzol and chloroform are hazardous chemicals and should only be handled in a well ventilated fume hood. Please read appropriate safety information before use.

6. RNA is very sensitive to degradation. Never touch any tubes, tips or surfaces that are to be used for RNA isolation with bare hands. Avoid unnecessary talking and breathing towards the samples while performing RNA extractions.

7. Always use filter tips during RNA isolation. Never reuse filter tips or use tips that have accidentally touched any contaminated surface.

8. All volumes in this protocol are based on tubes containing platelets dissolved in 1 mL TRIzol. If a platelet sample has been dissolved in more than 1 mL TRIzol, the sample will need to be subdivided into several tubes of 1 mL TRIzol each during the RNA purification. The resulting RNA pellets from the same donor can then be pooled into one tube during the RNA pellet ethanol wash step (section 3.4, step 12).

9. In order to avoid accidental sample loss and contamination of the RNA samples, clear RNAse free 1.5 mL tubes are preferred over large ultracentrifuge tubes for precipitation and washing of the RNA samples

10. Chloroform easily drips out of 1000 μL filter tips. To dispense chloroform, it is recommended to use either 100 or 200 μL tips.

11. A greater RNA yield can be achieved by precipitating the RNA at -20oC over night or at -80oC for one hour in the presence of 10 μg glycogen.

12. In our hands, reverse transcription using random hexamer primers has resulted in better cDNA yields than cDNA generated using oligo-dT primers.

13. There are many different platforms for qPCR to choose from. We have had good success with Qiagen’s QuantiFast SYBR Green kit and QuantiTect Primer assays using a Rotor-Gene 6000 PCR cycler. For an overview of other qPCR platforms such as TaqMan probes, please see http://en.wikipedia.org/wiki/Real-time_PCR.

14. Several different housekeeping genes are available for relative real-time-PCR, including GAPDH and ACTB that we have successfully used for platelet qPCR(1, 14).

15. For thermal cyclers other than Rotor-Gene 6000, see the QuantiFast SYBR Green PCR Kit manual for optimal cycling conditions.

16. Scaling down the total qPCR reaction volume from 25 μl to 10 μl without reducing the amount of cDNA added will result in a 60% reduction in QuantiFast SYBR Green cost and a 2.5 step lower Ct value for most qPCR reactions. Scaling down the reaction volume from 10 μl to 5 μl without reducing the amount of cDNA per reaction will further reduce the reagent cost but will provide slightly higher Ct values compared to the 10 μl reactions.

17. For most primer pairs, optimal linear quantification is achieved in the 17 to 30 Ct range (i.e. 17-30 PCR cycles). If a gene is detected later than 30 cycles, it may be difficult or impossible to accurately quantify the gene of interest due to the low levels of template available for the

qPCR reaction. On the other hand, if a gene is detected earlier than 20 cycles, we recommend that the template cDNA is diluted further in order to minimize waste of cDNA template (table 10).

Figure Legends Figure 1. Phase separation of TRIzol RNA samples after addition of chloroform. The clear aqueous phase (top) contains the RNA, the white interphase (middle) contains DNA and the pink phenol phase (bottom) contains protein. To avoid contamination of the RNA by DNA from the interphase it is recommended that only 85% of the aqueous phase is transferred to a new tube. Figure 2. Melt curve analysis following a real-time PCR run. A single peak (a) suggests that only one amplification product is generated whereas no single peak is present in the non-specific melt curve (b). Figure 3. Primer efficiency plots. Following quantification of serially diluted cDNA template (see Table 7), an optimal qPCR assay (a) will be fit by a line with a slope (k) close to -3.3, which will translate into a primer efficiency (E) of 2.0. b) A non-functional assay will give a slope which can be either greater than, or less than the ideal slope of -3.3, resulting in a primer efficiency considerably different from 2.0.

Acknowledgments The author would like to thank the Swedish Research Council and Prof. David Erlinge at Lund University, Sweden for their support. References

1. Amisten S, Braun OO, Bengtsson A, Erlinge D. Gene expression profiling for the identification of G-protein coupled receptors in human platelets. Thromb Res. 2008;122(1):47-57. 2. Bugert P, Dugrillon A, Gunaydin A, Eichler H, Kluter H. Messenger RNA profiling of human platelets by microarray hybridization. Thromb Haemost. 2003 Oct;90(4):738-48. 3. Gnatenko DV, Dunn JJ, McCorkle SR, Weissmann D, Perrotta PL, Bahou WF. Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood. 2003 Mar 15;101(6):2285-93. 4. McRedmond JP, Park SD, Reilly DF, Coppinger JA, Maguire PB, Shields DC, et al. Integration of proteomics and genomics in platelets: a profile of platelet proteins and platelet-specific genes. Mol Cell Proteomics. 2004 Feb;3(2):133-44. 5. Rox JM, Bugert P, Muller J, Schorr A, Hanfland P, Madlener K, et al. Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique. Clin Chem. 2004 Dec;50(12):2271-8. 6. Rolf N, Knoefler R, Suttorp M, Kluter H, Bugert P. Optimized procedure for platelet RNA profiling from blood samples with limited platelet numbers. Clin Chem. 2005 Jun;51(6):1078-80. 7. Available from: http://www.pall.com/medical_48187.asp. 8. RNeasy MinElute Cleanup Handbook2007. 9. Kendziorski C, Irizarry RA, Chen KS, Haag JD, Gould MN. On the utility of pooling biological samples in microarray experiments. Proc Natl Acad Sci U S A. 2005 Mar 22;102(12):4252-7. 10. Kendziorski CM, Zhang Y, Lan H, Attie AD. The efficiency of pooling mRNA in microarray experiments. Biostatistics. 2003 Jul;4(3):465-77. 11. Mary-Huard T, Daudin JJ, Baccini M, Biggeri A, Bar-Hen A. Biases induced by pooling samples in microarray experiments. Bioinformatics. 2007 Jul 1;23(13):i313-8. 12. Zhang W, Carriquiry A, Nettleton D, Dekkers JC. Pooling mRNA in microarray experiments and its effect on power. Bioinformatics. 2007 May 15;23(10):1217-24. 13. Wilson CL, Pepper SD, Hey Y, Miller CJ. Amplification protocols introduce systematic but reproducible errors into gene expression studies. Biotechniques. 2004 Mar;36(3):498-506. 14. McCloskey C, Jones S, Amisten S, Snowden RT, Kaczmarek LK, Erlinge D, et al. Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count. J Physiol. 2010 May 1;588(Pt 9):1399-406.

Table 1. Thermal cycling conditions for reverse transcription using the TaqMan reverse transcription kit.

Table 2. Preparation of master mix for cDNA synthesis. All volumes are in l. Table 3. Rotor-Gene 6000 cycling conditions for qPCR using the QuantiFast SYBR Green PCR Kit. Table 4. Preparation of master mix solution for pilot qPCR quantification using the QuantiFast SYBR Green PCR Kit. Table 5. Preparation of master mix for determination of primer assay efficiency. Table 6. Calculations of required amounts of cDNA for determination of primer amplification efficiency for the genes P2RY1, P2RY12 AND ACTB. Table 7. Volumes required for serial dilutions of cDNA for primer amplification efficiency determination. Table 8. Calculation of average Ct and dilution factor log values for the dilution curve of ACTB. Table 9. Preparation of mastermix for the quantification of one gene in duplicate reactions in at least three biological replicates, resulting in a master mix for 7 reactions ( 3 x 2 + 1 =7 reactions). Table 10. Potential of cDNA dilution in order to economize cDNA without compromising qPCR quality and accuracy. If the pilot qPCR returns Ct values lower than 20 to 25 cycles, it is possible to dilute the template cDNA without compromising qPCR quality and accuracy.

Table 1.

Temp C Time (minutes)

25 10

48 30

95 5

4 Eternity

Table 2.

Reaction volume 25 μL

Make for number of reactions 1x

10x TaqMan RT-buffer 2.50

25mM MgCl2 5.50

10 mM dNTP 5.00

Random hexamers 1.25

RNase inhibitor 0.50

MultiScribe Transcriptase 0.65

Master mix to each tube 15.40

RNA to each tube (60-2000 ng) 9.60

Table 3.

Step Temp (C) Time (sec) Flourescence acquisition

1 Initial denaturation 95 300 None

2 Cycling Denaturation 95 10 None

3 Cycling Annealing/Extension 60 30 Sybr/FAM

4 Repeat step 2-3 40 cycles

5 Melt curve 65-95* 5-30* Sybr/FAM

*Rising by 1 C each step, wait for 30 sec on first step, then wait for 5 sec for each step afterwards.

Table 4.

Number of 10 μL assays 1 x 10 μl 3.5 x 10 μl

Mastermix 5 17.5

Primer assay mix 0 0

Diluted cDNA 1 3.5

Volume mastermix added to each qPCR reaction 6 6

Volume QuantiTect Primer assay diluted 1 in 4 added to each qPCR reaction 4 4

Table 5.

Number of 10 μL assays 1 x 10 μl 17.5 x 10 μl

Mastermix 5 87.5

QuantiTect Primer assay 1 17.5

Volume mastermix added to each qPCR reaction 6 6

Volume diluted cDNA added to each qPCR reaction 4 4

Table 6.

Number of primer assays to test +1 4

Technical replicates 3

Diluted cDNA needed for each dilution factor step (number of assays x technical replicates) 12

Extra water to add so that 4 μl diluted cDNA can be added per assay* 36

*It is easier to accurately pipette 4μL than 1 μL, so extra water is added to the diluted cDNA to allow 4 μL to be added to each reaction tube.

Table 7.

Final cDNA dilution factor Volume cDNA and water added to each dilution step

1 6 μL of undiluted cDNA stock + 90 μL water

2 48 μL water + 48 μL cDNA from the above dilution step

4 48 μL water + 48 μL cDNA from the above dilution step

8 48 μL water + 48 μL cDNA from the above dilution step

16 48 μL water + 48 μL cDNA from the above dilution step

Table 8.

Dilution factor Log dilution factor Ct repl. 1 Ct repl. 2 Ct repl. 3 Average Ct

1 0.00 17.19 17.29 17.29 17.26

2 -0.30 18.38 18.42 18.20 18.33

4 -0.60 19.20 19.33 19.34 19.29

8 -0.90 20.30 20.37 20.35 20.34

16 -1.20 21.31 21.39 21.38 21.36

Table 9.

Number of 10 μL assays 1 x 10 μl 7 x 10 μl

Mastermix 5 35

Volume undiluted QuantiTect Primer assay 1 7

Volume mastermix added to each qPCR reaction 6 6

Volume diluted cDNA added to each qPCR reaction 4 4

Table 10.

detection Ct value (qPCR cycle number)

cDNA dilution factor to reach Ct 20

cDNA dilution factor to reach Ct 25

15 32 1024

16 16 512

17 8 256

18 4 128

19 2 64

20 1 32

21

16

22

8

23

4

24

2

25

1

Interphase with DNA

Aqueous phase with RNA

Phenol phase with protein

Transfer only 85% of aqueous phase to avoid DNA contamination

Figure 1

Specific melt curve

Figure 2a

Non-specific melt curve

Figure 2b

Figure 3.

a) b)

E=10(-1/3.39)=1.97 E=10(-1/1.32)=5.70

Ideal primer pair Poorly performing primer pair