RNA was extracted with the RNeasy
® Mini Kit (Qiagen, Hilden, Germany), followed by digestion of genomic DNA using the RNase free DNase-Set (Qiagen). RNA content was measured by spectrophotometry. The cDNA synthesis was conducted with SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen) using 1 μg of the extracted total RNA. The cDNA was analyzed in a multiplex analysis using TaqMan
® Universal PCR MasterMix and specific primers for SRD5A1 or the internal controls (Applied Biosystems, Foster City, CA, USA). The primers specific for SRD5A1 had the Assay ID: Hs00602694_mH, reporter dye FAM). Cyclophilin A served as the endogenous control and had the reference 4310883E (VIC/TAMRA Probe, Primer Limited). The reagents were assembled in MicroAmp™ Fast Optical 96-Well Reaction Plates (Applied Biosystems) and the PCR reaction was conducted in a 7500 Fast Real-Time PCR System (Applied Biosystems). The regular RT-PCR program was used and the read-out was set to automatic threshold (C
t). Each cell culture sample was measured in triplicates, non-template controls were included to detect potential contamination. In order to apply the ΔΔC
t method, a validation experiment according to the protocol provided by Applied Biosystems [
16] was conducted first, to demonstrate that multiplex analysis of SRD5A1 and cyclophilin was feasible. The validation experiment was successful with an absolute value of the slope of ΔC
t vs. log input of RNA being < 0.1 (-0,07, data not shown). The qRT-PCR protocol from Applied Biosystems [
15] and the publication by Yuan et al [
17] describe the ΔΔC
t-method used for analysis of the real-time PCR data. In brief, the cycle number at the C
t for the target gene (SRD5A1) was subtracted by the C
t of the reference gene (cyclophilin A) to give the ΔC
t value. The ΔC
t of the treated sample was then subtracted from the ΔC
t of the untreated sample to give ΔΔC
t. The exponential function of 2
-ΔΔCt represents the relative amount of the target gene in the treated sample compared to the target gene in the untreated control.
For the quantitative expression analysis of lung carcinoma and NAT samples obtained from patients, additional SRD5A1 primers were used (Assay ID Hs00971643_g1, reporter Dye FAM), as well as a second internal control, namely 18S rRNA (Reference 4319413E, VIC/MGB probe, Primer Limited). RNA and cDNA were prepared as before, and the regular RT-PCR program used to determine C
t values. Each sample value was obtained by the average of duplicate experiments. The mean C
t value (raw means and standard deviation) measured for the different primers is shown in Table
2. For statistical analysis, the repeated measures design (per patient: lung carcinoma and NAT, SRD5A1 and control primer pairs) was taken into account and a mixed linear model was used for fitting.
Table 2
Expression levels of SRD5A1 in NSCLC and NAT samples as measured by qRT-PCR.
SRD5A1 #1 | NAT | 9 | 30.74 | 0.97 |
| tumor | 23 | 29.25 | 1.37 |
SRD5A1 #2 | NAT | 9 | 31.30 | 1.16 |
| tumor | 23 | 29.45 | 1.69 |
Cyclophilin | NAT | 9 | 21.28 | 0.52 |
| tumor | 23 | 20.46 | 0.98 |
18S rRNA | NAT | 9 | 16.94 | 0.56 |
| tumor | 23 | 17.01 | 0.67 |