The document provides an overview of quantitative PCR (qPCR) and its applications. It discusses: 1. The basic steps of qPCR including RNA isolation, cDNA synthesis via reverse transcription, and quantitative PCR followed by statistical analysis of the results. 2. Different methods for RNA isolation from various samples like cells, tissues, and FFPE samples. 3. Primer and probe design considerations for qPCR and how to use probe design tools. 4. Details of the qPCR process like reaction composition, controls, and statistical tests used to analyze quantitative PCR results.

1. 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

2. 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

3. 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

4. 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

5. PRIMER DESIGN
 Choose sense primer
 Choose antisense primer
 Choose probe
 Or use TaqMan assay

6. 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.

7. WORKING SCHEME OF PROBES

9. 1. GO TO QPCR.PROBEFINDER.COM

10. 2. SELECT ORGANISM

11. 3. SPECIFY YOUR TARGET

12. 4. CHOOSE THE SEQUENCE

13. 5. ENTER YOUR EMAIL ADDRESS AND PRESS
“DESIGN”

14. 7. PROBEFINDER WILL DESIGN OPTIMAL
REAL-TIME PCR ASSAY

15. TAQMAN ASSAYS

16. 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...

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

18. 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

19. QPCR RESULTS

20. QPCR RESULTS

21. QPCR RESULTS

22. 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

23. STATISTICAL ANALYSIS