REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION (Q-PCR)

 

Attila Szanto

 

University of Debrecen, Medical and Health Science Center, Department of Biochemistry and Molecular Biology, Life Science Building, Room 3-211, Egyetem ter 1, Debrecen, H-4010, Hungary, Tel.: +36 52 416432, Fax: +36 52 314989

szantoa@indi.biochem.dote.hu

 

 

INTRODUCTION TO THE PROCEDURE

 

Real-time quantitative polymerase chain reaction (Q-PCR) is the most sensitive method for detection and quantification of RNA or DNA molecules. It is a complex method that requires expertise in PCR assay development and optimization. Nevertheless, it is a powerful method with high specificity, sensitivity and reproducibility that can be used in high-throughput studies. Basically, Q-PCR is a fluorescence-based kinetic study where fluorescent signal produced by the proceeding amplification of a specific template is determined in every PCR cycle. The real time determination of the fluorescent signal makes it possible to use the log phase of the PCR for quantification, when the signal correlates with the initial number of template molecules, and at the same time provides high sensitivity when comparing small differences. Another advantage of the technique is that molecules with low-abundancy can also be quantified precisely, allowing the analysis of limiting amounts of cells or tissues.

 

Reverse transcription

 

For the analysis of the transcriptome we need quantification of the expressed RNA molecules. There are five commonly used methods for this: Northern-blotting, RNase protection assays, in situ hybridization, microarrays and reverse transcription (RT)-PCR. Among these real time RT-Q-PCR is the most sensitive and suitable method for quantification. To analyze RNA molecules with Q-PCR, they need to be transcribed into cDNA molecules by reverse transcriptase. This can be primed by different methods: gene-specific primers, random primers or oligo-dT primers.

 

Quantification of cDNA/DNA molecules

 

Quantitative information is generated from fluorescent signal that correlates with the initial copy number of cDNA or DNA molecules. This signal can be generated from non-specific binding of a fluorescent dye to the amplified nucleic acid (e.g. SYBR Green I) or from sequence specific, fluorescently labeled oligonucleotides, also called probes (e.g. Taqman probes). Both methods can quantify the mRNA/DNA copy numbers of the gene-of-interest, either in an absolute manner when using standard curves (serial dilution of an amplicon with know copy numbers), or in a relative manner when comparing to a specific sample. In both cases normalization to A) a consitutively expressed and not regulated mRNA or B) a gene with stable genomic copy number is essential.

 

 

 

OUTLINE OF THE EXPERIMENTS

 

 

In these experiments we will analyze the effects of DMSO priming and 9-cis retinoic acid treatment of CDM1 cells on different levels. Previous experiments are analyzed with one common method, Q-PCR in the following manner:

 

 

 

 


REAL-TIME QUANTITATIVE PCR ANALYSIS - PRACTICALS

 

 

Practical 8 – Quantification of ChIP DNA

 

Each group will analyze four samples:

            Input

            No Antibody Control (NAB)

            H4Ac

            H3K4Met

 

Four genes will be measured from every sample:

 

                        Transglutaminase 2 core promoter (Core)

                        Transglutaminase enhancer HR1 (HR1)

                        CD38 Retinoic Acid Receptor Response Element (CD38)

                        36B4 (intron-less gene for control)

 

Q-PCR analysis will be performed in 384 well plates with a pipetting robot.

 

 

 

Practical 8A: Real-time PCR with TaqMan probes

 

Checklist:

            Samples in strips:       Input

                                                NAB

                                                H4Ac

                                                H3K4Met

            2x Taqman Master Mix

            Primers and Probes

 

 

 

DNA samples will be measured in triplicates. Six groups will use one plate. Prepare DNA samples and PCR Master mixes according to Figure 1. Note, that six groups prepare four PCR Master mixes.

 

 

 

 

 

 

 

 

                                                            (For one reaction)                   For 100 reactions

 

2x Taqman Master Mix                       5 ml                                          500 m

Forward primer (100 mM)                  0.0375 ml                                 3.75 ml

Reverse primer (100 mM)                   0.0375 ml                                 3.75 ml

Probe (20 mM)                                                0.0625 ml                                 6.25 ml

Final Volume (approximately)            5 ml                                          500 ml

 

 

 


 


When both the samples and the PCR master mixes are located in the correct position the robot will be started.

 

When the plate is finished cover it with adhesive optical cover and spin it in the plate centrifuge. Then load plate into the Q-PCR (AB 7900) machine or leave it on ice till starting the PCR.

 

           

 

 

PCR cycle:      1. 95oC                        10 min

                                    2. 95oC                        15 sec

                                    3. 60oC                        60 sec

                                    Repeat Steps 2 and 3 40 times

 


Practical 9 – Quantification of mRNA

 

 

Practical 9A: Establishing Standard Curves

 

Checklist:

            Transglutaminase amplicon (6e7)

            Marked Eppendorf tubes

            Yeast tRNA

            2x Taqman Master Mix

            Primers and Probe

            Nuclease-free water

 

1. Make a serial dilution of Transglutaminase 2 amplicon.

            6e7

            6e6

            6e5

            6e4

            6e3

            6e2

            6e1

 

Repeat these steps with the other tubes (6e5 to 6e1), pipetting Tg amplicon from the dilution made in the previous step to obtain all the 7 log dilution series of the amplicon.

 

2. Prepare PCR Master mix for Transglutaminase for 30 reactions:

 

                                                            (For one reaction)                   For 30 reactions

 

2x Taqman Master Mix                       10 ml                                        300 m

Forward primer (100 mM)                  0.075 ml                                   2.25 ml

Reverse primer (100 mM)                   0.075 ml                                   2.25 ml

Probe (20 mM)                                                0.125 ml                                   3.75 ml

Nuclease-free water                             7.725 ml                                   231.75 ml

Final Volume                                      18 ml

 

 

3. Fill up the 96-well PCR plate:

 

Samples will be measured in triplicates. Similarly, No Template Control (NTC) will be measured in triplicates as indicated in Figure 2. Note that 4 groups will use one plate.

 


 

 

 

 

 

            PCR cycle:      1. 95oC            10 min

                                    2. 95oC            12 s

                                    3. 60oC            30 s

                                    Repeat Steps 2 and 3 40 times

 


Practical 9B: Reverse Transcription

 

The same RNA used for the microarray experiments will be analyzed in this experiment. Note that a portion of the RNA prepared with the Qiagen RNeasy kit was DNase-treated prior to this experiment.

 

Checklist:

            RNA sample from Practical 1

            5x First Strand Buffer

            dNTP (2.5 mM)

            DTT (0.1 M)

            Nuclease-free water

            Random primer

            Reverse Transcriptase will be provided by the assistance

 

 

 

For one reaction

RT                               No-RT

 

5x First Strand Buffer                                     8 ml                              3 ml

dNTP (2.5 mM)                                              8 ml                              3 ml

DTT (0.1 M)                                                   4 ml                              1.5 ml

Random Primer (1 mg/ml)                                1.2 ml                           0.45 ml

Reverse Transcriptase (200 U/ml)                   0.4 ml                           -

Final Volume                                                  21.6 ml                         7.95 ml

 

Add DNase-treated total RNA (150 ng/ml)     18.4 ml                         6.9 ml

Nuclease-free water                                         -                                   0.15 ml

Final Volume                                                  40 ml                            15 ml

 

 

            PCR cycle:      25oC    10 min

                                    42oC    2 h

                                    72oC    5 min

                                    4oC      hold

 

cDNA samples can be stored at -20oC.


Practical 9C: Real-time PCR with TaqMan probes

 

 

Every group will measure four genes: Cyclophylin

                                                                        36B4

                                                                        Transglutaminase 2 (Tgm2)

                                                                        CD38

 

Checklist:

            cDNA samples (RT and No-RT) from Practical 9B

            2x TaqMan Master Mix

            Nuclease-free water

            Forward, reverse primers and Probes

 

 

  1. Prepare PCR Master mixes for five reactions (one for each gene being measured)

 

      Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

 

                                                            (For one reaction)                   For 5 reactions

 

2x Taqman Master Mix                       10 ml                                        50 ml   

Forward primer (10 mM)                    0.75 ml                                     3.75 ml

Reverse primer (10 mM)                     0.75 ml                                     3.75 ml

Probe (10 mM)                                                0.25 ml                                     1.25 ml

Nuclease-free water                             3.25 ml                                     16.25 ml

Final Volume                                      15 ml

 

      Mix by vortexing, then spin down briefly in centrifuge

 

2. Fill up the 96-well PCR plate:

RT samples will be measured in triplicates while No-RT control will be measured in a single well. Six groups will use one plate.

 

      Pipette 15 ml PCR Master mix into 4 wells of a 96-well plate as indicated in Figure 3.

      Pipette 5 ml of cDNA (RT) into the first 3 wells and 5 ml sample (No-RT) into the 4th well.

      Repeat these with the other genes as indicated in Figure 3.

 

      When the plate is finished cover it with adhesive optical cover and spin it in plate centrifuge then load plate into the Q-PCR (AB 7900) machine or leave it on ice till starting the PCR.

 

            PCR cycle:      1. 95oC                        10 min

                                    2. 95oC                        12 sec

                                    3. 60oC                        30 sec

                                    Repeat Steps 2 and 3 40 times

 


 


Alternative protocol for the PCR, if not using a commercial master mix:

 

                                                            For one reaction

10x PCR Buffer                                   2 ml

MgCl2 (25 mM)                                   2.4 ml

dNTP (2.5 mM)                                   1 ml

Forward primer (100 mM)                 0.75 ml

Reverse primer (100 mM)                   0.75 ml

Probe (20 mM)                                    0.125 ml

Taq polymerase (5 U/ml)                    0.125 ml

ROX reference (50x)                           0.4 ml

Nuclease-free water                            7.45 ml

Final Volume                                       15 ml

 

cDNA                                                  5 ml

Final Volume                                       20 ml


Practical 10 – Quantification of miRNA

 

 

Practical 10A: Reverse Transcription

 

We are using High Capacity cDNA Archive Kit (Applied Biosystems) to transcribe miRNAs from Trizol-purified total RNA samples. Note that Qiagen preps cannot be used for miRNA analysis because small RNA molecules are lost during that procedure while they are kept during Trizol purification.

 

The following miRNAs will be measured:      hsa-miR-34a

                                                                        hsa-let-7i

                                                                        hsa-miR-338

                                                                        hsa-let-7a

RNU43 (U43 small nucleolar RNA)

 

Each group will measure two miRNAs and one normalizer small nucleolar RNA:

 

            Group 1, 4, 7 and 10:  hsa-miR-34a

                                                hsa-let-7a

                                                RNU43

            Group 2, 5, 8 and 11:  hsa-miR-7i

                                                hsa-let-7a

                                                RNU43

            Group 3, 6, 9 and 12:  hsa-miR-338

                                                hsa-let-7a

                                                RNU43

 

Checklist:

            Trizol-isolated RNA samples from Practical 2

            10xRT Buffer

            dNTP (10 mM)

            Nuclease-free water

            Reverse Transcriptase, RNAsin and 5xRT Primer will be provided by the     assistance

 

 

 

 

 

 

 

 

 

 

 

1. Prepare RT master mix for four reactions in a 1.5 ml Eppendorf tube on ice:

 

      Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

 

 

RT 1-3

(For one reaction)                   For 4 reactions

 

10xRT Buffer                                      1.2 ml                                       4.8 ml

dNTP (10 mM)                                   1.2 ml                                       4.8 ml

Reverse Transcriptase (50 U/ml)         0.8 ml                                       3.2 ml

RNAsin (40 U/ml)                               0.15 ml                                     0.6 ml

Nuclease-free water                             4.25 ml                                     17 ml

 

Final Volume                                      7.6 ml                                       30.4 ml

 

That will give you a final volume of 12 ml.

 

 

2. Prepare the following No-RT master mix for four reactions in a 1.5 ml Eppendorf tube on ice.

 

               Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

No-RT 1-3

(For one reaction)                   For 4 reactions

 

10x RT Buffer                                     1.2 ml                                       4.8 ml

dNTP (10 mM)                                   1.2 ml                                       4.8 ml

RNAsin (40 U/ml)                               0.15 ml                                     0.6 ml

Nuclease-free water                             5.05 ml                                     20.2 ml

 

Final Volume                                      7.6 ml                                       30.4 ml

 

That will give you a final volume of 12 ml.

            PCR cycle:      16oC    30 min

                                    42oC    30 min

                                    85oC    5 min

                                    4oC      hold

Tubes can be stored at -20oC.


Practical 10B: Real-time PCR with TaqMan probes

 

Checklist:

            Reverse transcribed miRNA samples from Practical 10A

            2x TaqMan Master Mix

            Nuclease-free water

            10x TaqMan miRNA assays will be provided by the assistance

 

 

RT samples will be measured in triplicates while No-RT control will be measured in a single well. 6 groups will use one plate. See Figure 4.

 

1. Prepare PCR Master mixes 1, 2, 3 (one for each miRNA being measured), respectively.

 

      Mix the following components in a 1.5 ml Eppendorf tube, on ice:

 

                                                            (For one reaction)                   For 5 reactions

 

2x Taqman Master Mix                       7.5 ml                                       37.5 ml

10x Taqman miRNA assay                 1.5 ml                                       7.5 ml

Nuclease-free water                             4.5 ml                                       22.5 ml

 

Final Volume                                      13.5 ml                                     67.5 ml

 

      Mix

 


 

 


      Pipette 13.5 ml PCR Master mix into 4 wells of a 96 well plate as indicated in Figure 3.

      Pipette 1.5 ml of RT Product (RT) into the first 3 wells and 1.5 ml RT Product (No-RT) into the 4th well as indicated in Figure 4.

      When the plate is finished cover it with adhesive optical cover and spin it in plate centrifuge then load plate into the Q-PCR (AB 7900) machine or leave it on ice till starting the PCR.

 

            PCR cycle:      1. 95oC            10 min

                                    2. 95oC            15 s

                                    3. 60oC            60 s

                                    Repeat Steps 2 and 3 40 times

 

 


Tips and tricks

 

 

 

 

 

 

 

 

 

 


Reagents

 

2x SYBR Green Master Mix – Diagenode – GMO-SG2x-A300

2x Taqman Master Mix – Diagenode – GMO-MM2x-A300

Yeast Transfer RNA 25 mg – Invitrogen – 15401-011

Primers – Sigma-Genosys

SuperScript II RNase H-Reverse Transcriptase, 10000U, 200U/ml – Invitrogen – 18064-014

5xFirst Strand Buffer – Invitrogen – Y00146

DTT, 0.1 mM, 500 ml – Invitrogen – Y00147

Random Primer, 300 mg, 3 mg/ml – Invitrogen – 48190-011

20x Reference Dye, 500 ml – Invitrogen – 54881

dATP, 100 mM, 25 mmol – Fermentas – R0141

dCTP, 100 mM, 25 mmol – Fermentas – R0151

dGTP, 100 mM, 25 mmol – Fermentas – R0161

dTTP, 100 mM, 25 mmol – Fermentas – R0171

RiboLock Ribonuclease Inhibitor, 40 U/ml, 2500U – Fermentas – EO0381

Taq DNA Polymerase, 5 U/ml, 500U – Fermentas – EP0402

miRNA Assays – Applied Biosystems

cDNA Archive Kit, 200 rxn, 100 ml – Applied Biosystems – 4322171