Quantitative RT-PCR analysis

CELL TREATMENT

H9 hES cells treated with 10 µM thalidomide or DMSO for 24 h in biological duplicates were subjected to gene expression analysis.

RNA ISOLATION

RNA was isolated using the RNeasy Plus mini kit (Qiagen).

REVERSE TRANSCRIPTION

cDNA was created by reverse transcription using ProtoScript II reverse transcriptase (NEB; Catalog #M0368) following the manufacturer's instructions.

The following primer sets from IDT were used with SYBR Green Master Mix (Applied Biosystems; Catalog #A25779) to probe both GAPDH and total SALL4 levels: 

SALL4total – F: GGTCCTCGAGCAGATCTTGT

SALL4total – R: GGCATCCAGAGACAGACCTT 

GAPDH – F: GAAGGTGAAGGTCGGAGTC 

GAPDH – R: GAAGATGGTGATGGGATTTC

Set up a RT-PCR reaction of 800 ng of RNA in total volume of 8 µL as follows. 

Step 1:

Mix the following and incubate at 25°C for 5 min followed by incubation on ice.

Step 2:

Add the following to the mix in step 1 and incubate at 42°C for 60 min, followed by incubation at 65°C for 20 min.

Resulting cDNA is diluted 1:10 to 5 ng/µL in preparation for the qPCR reaction.

qPCR

Use 8 µL of the cDNA at 5 ng/µL to provide 40 ng cDNA per qPCR reaction.

Prepared 14 reactions total to allow biological duplicates and technical replicates, plate layout was as follows:

1. Prepare qPCR mix for each sample reaction following the protocol below:

2. 20 µL of each biological replicate set was dispensed into three wells of a 96-well white plate to create technical replicates.

3. The plate was sealed with transparent film and centrifuged for 1 min at 500 xg.

4. A CFX Connect Real-Time PCR System (Bio-Rad) was used to quantify expression changes using the following thermal cycling parameters:

DATA ANALYSIS

The fold change in gene expression normalized to a house keeping gene and relative to untreated control was calculated using the 2^-ΔΔCT method (Livak and Schmittgen, 2001).

To do this:

1. The raw Ct values for the replicates of GAPDH and SALL4 were averaged to find the average Ct for GAPDH and SALL4 in each of the two treatment groups (thalidomide treated and control).
2. The SALL4 Ct was normalized to GAPDH reference in each treatment group to provide the ΔCT for DMSO and Thalidomide treated samples.
3. The difference between treated and control samples was calculated to find the ΔΔCT (double delta Ct) value.
4. The 2^-ΔΔCt was then calculated to indicate the expression fold change.

REFERENCES

Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods 25, 402-408.

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