Primescript 1st strand complementary dna synthesis kit

Tissue specimens were grounded and added with TRIzol reagent (Takara). Then the total RNA was isolated, and 1 μg of RNA was reverse-transcripted with PrimeScript 1st Strand complementary DNA Synthesis kit (Takara). Real-time PCR assay was performed on ABI 7500 platform. SYBR Premix Dimer Eraser kit (Takara) was used in 20 μl reaction volume, and the cycling conditions were as follows: an initial 30 s denaturation at 95°C and 45 cycles (5 s at 95°C, 30 s at 55°C, and 34 s at 72°C). PPIA and B2M genes were set as internal controls. MCM2, RNASEH2A, and TOP2A expression level was detected in sixteen pairs of colorectal cancer and adjacent mucosa samples. The primer sequences were shown in Supplementary Table
1.

Briefly, tissue specimens were lysed and homogenized in the presence of a highly denaturing guanidine-thiocyanate-containing buffer that inactivates RNases. Trizol reagent (Sigma, USA) added to provide appropriate binding conditions were grounded. Then, the total RNA was isolated based on the RNAeasy Mini Kit (Qiagen Cat No. /ID: 74,104) protocol. One μg of RNA was reverse transcripted with PrimeScript 1st Strand complementary DNA Synthesis kit (Takara). Real-time PCR assay was performed on the Qiagen Rotor Gen Q system using the SYBR green Premix Dimer Eraser kit (TaKaRa cat number: RR820Q). The cycling conditions were an initial 30 s denaturation at 95 °C and 40 cycles (5 s at 95 °C, 30 s at 60 °C, and 45 s at 72 °C). The GAPDH gene was set as internal control; Talin1 expression level was detected in forty pairs of CRC and adjacent normal tissue samples. The primer sequences were as follows: GAPDH: 5′-AACTTTGGCATTGTGGAAGG-3′ F and 5′-CACATTGGGGGTAGGAACAC-3′ R. Talin1: 5′-.TTGGAGATGCCAGCAAGCGACT-3′ F and 5′-CCAGTTCTGTGGCTGCCTGATT-3′. The expression levels of Talin1 mRNA were normalized against GAPDH levels based on the 2^−ΔΔCt approach^{77 (link)}.