Quantitative PCR

This protocol automates quantitative PCR (qPCR) for real time DNA detection and quantitation on Transcriptic’s robotic platform.

OVERVIEW

Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a common fluorescence-based laboratory technique used in gene expression studies to determine the absolute or relative quantities of DNA in a sample in real-time. There are 2 common techniques used characterized by the different underlying chemistries: 1) non-specific detection using fluorophores such as SYBR Green, that intercalate and bind to double-stranded DNA (dsDNA) and (DNA-Binding Dye Assays) 2) a DNA sequence-specific fluorescent probe consisting of a dye reporter and quencher as readout for template DNA quantity (Probe-Based Assays).

DNA-BINDING DYE ASSAY

Non-specific fluorophores such as SYBR Green (excitation and emission of 494 nm and 521 nm, respectively) bind dsDNA and increase in fluorescence output can be captured and monitored using a qPCR device. During PCR amplification, the fluorescence emitted is directly proportional to the increase in product. Advantages of this method include ease of use, convenience and cost-effectiveness as only primers targeting the intended sequence and template DNA are required in the reaction mixture. However, SYBR binds nonspecifically to all dsDNA products produced in the PCR reaction including undesirable by-products such as primer dimers, which can heavily interfere with and bias accurate quantitation of the desired target. Also, this method is not amenable to multiplexing as only 1 target sequence can be monitored per reaction.

METHODOLOGY

The DNA-binding dye assay was executed and validated on the Transcriptic platform using a 5-point 10-fold serial dilution of a purified plasmid DNA (pDNA) starting with a concentration of 1 ng/ul. Reactions were assembled, 2.5 microliters total volume, in a 384-well pcr plate using an Echo 525 acoustic liquid handler (Labcyte). Amplification was achieved using a SensiFASTTM SYBR No-ROX kit from Bioline (Cat No. BIO-98005) with primers from Integrated DNA Technologies targeting a 265 base pair amplicon on the pDNA. Samples were assayed in 64 replicates on the Bio-Rad CFX384 Touch Real-Time PCR Detection system. For information on amplification for other sample input types as well as custom reagents, please contact [email protected]

RESULTS

10-fold serial dilutions of plasmid DNA (starting concentration 1 ng/ul) were assayed in 64 replicates using a SensiFASTTM SYBR No-ROX kit from Bioline (Cat No. BIO-98005) with primers from Integrated DNA Technologies targeting a 265 base pair amplicon. DNA amplification on the Bio-Rad CFX384 Touch Real-Time PCR Detection system is plotted below as relative fluorescent units vs. cycle number (Figure 1).

Figure 1.Amplification of DNA depicted as relative fluorescence units (RFU) vs. cycle number.

Statistical analysis of CT data, standard deviations (S.D.), percentage coefficient of variance (%CV), and linear regression was performed, see Figure 2 below.

Figure 2.Statistical analysis of technical replicates and linear regression over the 10-fold serial dilutions.

Probe-Based Assay

Probe-based qPCR uses fluorescent reporter probes that monitor and detect changes in sequence-specific products. Unlike SYBR Green which binds to all dsDNA, this strategy uses sequence-specific probes for improved assay sensitivity and specificity. Furthermore, a variety of different fluorophores allow for multiplexing and detection of multiple products within one reaction. In this method, a reaction is prepared similarly to the DNA-binding dye method described above but with the addition of a sequence-specific fluorescent probe that when intact, remains in a quenched state. The fluorophore is covalently attached to the 5’ end of the probe with a quencher at the 3’ end. During the annealing stage of PCR, both the reporter probe and primers bind to the target. Once the polymerase reaches the probe, the 5’ to 3’ exonuclease activity of the polymerase degrades the probe to release the fluorophore from its quenched state. Emitted fluorescence detected by the qPCR device is directly proportional to the starting amount of template and monitored in real-time during each cycle of PCR amplification.

Methodology

RNA was extracted from mouse liver tissue using the Thermo Scientific MagJET RNA Extraction kit (Cat No. K2731) on the KingFisher Flex Purification System. Concentration and purity were determined using a NanoDropLite Spectrophotometer. RNA samples were normalized, followed by reverse transcription (RT) using the SensiFASTTM cDNA Synthesis kit (Cat No. BIO-65053).

Reactions were assembled, 5 microliters total volume, in a 384-well pcr plate using an Echo 525 acoustic liquid handler. Amplification was achieved using a SensiFASTTM Probe No-ROX kit (Cat No. BIO-86005) and primer/probes from Life Technologies ThermoFisher. Samples were assayed in triplicate for the gene of interest and two endogenous controls. Reactions were performed on the Bio-Rad CFX384 Touch Real-Time PCR Detection system. For information on amplification for other sample input types as well as custom reagents, please contact [email protected]

Amplification of endogenous controls was performed to standardize the amount of sample DNA added to each reaction. The ∆CT value was determined by subtracting the average endogenous CT value (over technical replicates) from the average target of interest CT value. Relative expression of each gene of interest was determined using the comparative CT method (calculations for the method shown in Appendix I).

Results

Below are representative curves of the endogenous control and a target of interest. Using the relative expression method, quantitation of the target gene is performed on each sample. Mean group CTs and CT are calculated to determine experimental group effects.

Figure 3.Amplification of DNA depicted as relative fluorescence units (RFU) vs. cycle number. (A) FAM-labelled amplification monitoring endogenous control 1. (B) VIC-labelled amplification of target genes and endogenous control 2 (single-plex reactions).

Statistical analysis of CT data, standard deviations (S.D.), and percentage coefficient of variance (%CV) was performed on technical replicates, see Appendix II. Representative group ∆CT values are shown in the following figure showing separation between the Vehicle Control group, the Experimental Response group, and the Positive Control group.

Figure 3.Delta CTs calculated for Vehicle Control (Group 1), Experimental Response (Group 2), and Positive Control (Group 3) using Endogenous Control 1 and the Target Gene.

Platform capabilities are not limited to the above highlighted assays. Amplification for a variety of sample inputs types, as well as, use of custom reagents can be accommodated. For additional information, please contact [email protected].

Appendix I

The amount of target, normalized to an endogenous reference and relative to a calibrator, is given by:

where:

and:

Note: For the relative expression method, CT, to be valid, the efficiency of the target amplification and the efficiency of the reference amplification must be approximately equal.

qPCR capabilities on the Transcriptic Platform are not limited to the above highlighted assays. Please contact [email protected] for more information amplification using a wide variety of sample input types or custom reagents.

Appendix II

Statistical analysis of CT data, standard deviations (S.D.), and percentage coefficient of variance (%CV) was performed on technical replicates for the probe-based assay data shown in this report.

Group

Sample ID

Mean CT

SD CT

%CV CT

Mean HK1

S.D. HK1

%CV HK1

Mean HK2

S.D. HK2

%CV HK2

1

1

27.58

0.10

0.37

16.24

0.10

0.64

18.89

0.23

1.22

1

2

27.34

0.05

0.17

16.58

0.06

0.35

18.86

0.15

0.81

1

3

27.66

0.27

0.98

16.65

0.14

0.84

18.77

0.39

2.07

1

4

26.26

0.08

0.32

16.59

0.07

0.42

18.74

0.13

0.69

1

5

26.67

0.07

0.25

16.66

0.07

0.42

18.78

0.15

0.81

1

6

27.54

0.05

0.17

16.45

0.05

0.28

18.54

0.09

0.50

1

7

27.11

0.17

0.63

16.37

0.04

0.23

18.44

0.06

0.33

1

8

25.28

0.08

0.32

16.97

0.14

0.85

18.72

0.29

1.53

2

9

24.58

0.03

0.14

15.81

0.06

0.35

19.20

0.23

1.22

2

10

24.21

0.17

0.69

15.79

0.16

0.98

18.39

0.11

0.57

2

11

24.05

0.19

0.78

15.88

0.22

1.37

18.34

0.62

3.36

2

12

24.08

0.09

0.36

16.19

0.11

0.68

18.65

0.11

0.59

2

13

23.73

0.11

0.46

15.82

0.09

0.58

18.43

0.25

1.38

2

14

24.13

0.04

0.18

15.73

0.09

0.59

18.33

0.18

0.96

2

15

24.01

0.05

0.20

15.26

0.09

0.58

18.42

0.31

1.69

2

16

24.32

0.03

0.12

15.76

0.18

1.14

18.65

0.18

0.99

3

17

25.42

0.18

0.71

15.63

0.16

1.03

18.81

0.05

0.27

3

18

25.69

0.16

0.63

15.60

0.12

0.78

18.62

0.08

0.44

3

19

25.07

0.08

0.32

16.12

0.09

0.55

19.21

0.40

2.07

3

20

24.05

0.26

1.07

16.87

0.09

0.51

18.98

0.06

0.30

3

21

24.08

0.04

0.18

16.98

0.09

0.54

19.08

0.06

0.32

3

22

24.55

0.08

0.33

16.42

0.15

0.92

18.81

0.16

0.87

3

23

24.65

0.06

0.23

16.58

0.04

0.26

18.85

0.17

0.92