PRACTICAL COURSE ON QRT-PCR

Course of Lectures for young scientists

“Molecular and Cellular Bases of Host-Microbes Interactions: Genomics and

Sequencing”

5th June 2014

Hovsep Ghazaryan

GENE EXPRESSION ANALYZES

1. Isolation of RNA from cells / tissue / FFPE

2. Synthesis of cDNA (reverse transcription)

3. Quantitive Polymerase Chain Reaction

4. Statistical analyzes

RNA ISOLATION METHODS

1. Guanidinium thyocyanate-phenol-chlorophorm method

2. Column purification

A. RNA extraction from cells

B. RNA extraction from whole blood

C. RNA extraction from tissues

D. RNA extraction from formalin-fixed, paraffin-embedded tissue samples

REVERSE TRANSCRIPTION

65C for 5min, chill on ice

Total RNA (0.1-5 µg) 1 µl

Oligo dT primer 1 µl

Nuclease free water 9 µl

37C for 60min, 70C for 5min

MIX 11 µl

5X reaction buffer 4 µl

RNase inhibitor (20 u/µl) 1 µl

10 mM dNTP mix 2 µl

Reverse transcriptase (20 u/µl) 2 µl

PRIMER DESIGN

Choose sense primer

Choose antisense primer

Choose probe

Or use TaqMan assay

PROBES

Probes are short fragments of DNA of RNA.

The probes are used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe.

The probes are labeled with molecular marker of fluorescent or radioactive (in the past) molecules.

WORKING SCHEME OF PROBES

1. GO TO QPCR.PROBEFINDER.COM

2. SELECT ORGANISM

3. SPECIFY YOUR TARGET

4. CHOOSE THE SEQUENCE

5. ENTER YOUR EMAIL ADDRESS AND PRESS “DESIGN”

7. PROBEFINDER WILL DESIGN OPTIMAL REAL-TIME PCR ASSAY

TAQMAN ASSAYS

CHOOSE HOUSEKEEPING GENE

Housekeeping genes have constant

expression under normal and patho-

physologicial conditions

For each type of cells / tissues there are

different housekeeping genes

Examples: GAPDH, HSP90, β-actin, ACTB,

PSMB1, PSMB2...

CALLIBRATION

Take reference cDNA for callibration with different concentrations: 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1.25 μg/ml

QPCR

1 cycle 94C for 15 min, 40 cycles (94 C for 45 s, 60C for 30 s)

dH2O 7.8 µl

10X buffer 2.5 µl

25 mM MgCl2 3.5 µl

10 mM dNTPs 1 µl

Primer-L (10 pM/µl) 2.25 µl

Primer-R (10 pM/µl) 2.25 µl

Probe 0.5 µl

Taq polymerase 0.2 µl

QPCR RESULTS

QPCR RESULTS

QPCR RESULTS

STATISTICAL ANALYSIS

Check normal distribution with Kolmogorov-Smirnov test

If passes normality test, use Student’s T-test

For nonparametric distribution use Mann-Whitney U-test

STATISTICAL ANALYSIS