Rt2 qpcr primer assay for human gapdh

HEK 293T cells sorted as described in ‘Flow Cytometry’ below were counted using the Countess II Cell Counter (Thermo Fisher Scientific) and cell samples were diluted such that all conditions contained the same cell numbers as input. RNA was isolated using the Zymo DirectZol RNA MiniPrep Plus extraction kit with an on-column DNaseI digestion. RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcriptase kit (Applied Biosystems). Quantitative PCR was performed using SYBR green qPCR Master Mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems) with ReadyMade PrimeTime primers for SPI1 (Integrated DNA Technologies Inc, USA) and RT2 qPCR Primer Assay for Human GAPDH (cat# PPH00150F-200, Qiagen). Expression was quantified using ABI Sequence Detection software compared to serial dilutions of an SPI1 or GAPDH synthetic sequence gBlock (Integrated DNA Technologies Inc, USA). Measured values for SPI1 were normalized to measured values of GAPDH.

Total RNA from MDA231 and MDA231-BrM2 cells was extracted using the miRNeasy Micro Kit (cat# 217084, Qiagen, Germany). A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) was used to evaluate RNA purity and concentration using two absorbance ratios (A260/A280 and A260/A230). For single-stranded cDNA synthesis, the RT² First Strand Kit (cat#, 330401, Qiagen) or the miScript II RT Kit (cat#, 218161, Qiagen) was used as per the manufacturer’s protocols. Real-time quantitative PCR was performed on the “Applied Biosystems^® StepOne™ Real-Time PCR System” with the RT² SYBR Green ROX qPCR Mastermix (cat# 330522, Qiagen) for mRNA expression and the miScript SYBR Green PCR Kit (Cat# 218075, Qiagen) for miRNA expression using the “Applied Biosystems^® StepOne™ Real-Time PCR System”.13 (link) hsa-miR-623 and MMP1 relative expressions were normalized to the housekeeping gene U6 small nuclear RNA and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), respectively, and the data were analyzed using the 2^–ΔCt and 2^–ΔΔCt methods.34 (link) The primers used were obtained from Qiagen: Hs_miR-623_1 miScript Primer Assay (Qiagen), Hs_RNU6-2_11 miScript Primer Assay (cat# MS00033740), RT² qPCR Primer Assay for Human MMP1 (cat# PPH00120B-200), and RT² qPCR Primer Assay for Human GAPDH (Cat# PPH72843A).