Sybr green real time pcr

BM cell culture was harvested at day 3 and lineage-depleted. Briefly, cells were pelleted and incubated with biotin-lineage antibody cocktail (10 µl/10⁷ cells) (Miltenyi Biotec), followed by anti-biotin MicroBeads (Miltenyi Biotec). Cells were negatively selected using LS column as described. Lineage-negative cells were collected and preserved in TRIzol (Invitrogen, Victoria, Australia) at -80°C. Total RNA was isolated. RNA (1 µg) was reverse transcribed using random primers (Promega, Madison, WI) and M-MLV Reverse Transcriptase (Promega). cDNA (50 ng) was used as a template for SYBR Green real-time PCR (Roche, NSW, Australia) on a CFX96 Touch System (Biorad, Hercules, CA) using a standard three-step melt program (95°C for 15 s, 55°C for 30 s and 72°C for 30 s). Primers were designed for the quantification of C/EBPA (F 5 CGG TGC GCA AGA GCC GAG AT; R 5 CCC GCA GCG TGT CCA GTT CA) and c-fos (F 5 ACT AGA GAC GGA CAG ATC TG; R 5 ATA ACG GGA ACG CAG CAG TA). Data were normalized to the housekeeping gene HPRT1 (QuantiTec, Qiagen, Victoria, Australia) and presented as the relative level of expression.

To measure the mitochondrial DNA content, we determined the ratio of one mitochondrial gene to a nuclear gene using quantitative real-time RT-PCR as described previously with some modifications (Pieters et al., 2013 (link)). Total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's instructions (Quick-Start Protocol). The concentration of the extracted DNA was measured spectrophotometrically at 260 nm with the NanoDrop 2000 (Thermo Scientific, Wohlen, Switzerland). Afterwards, DNA was diluted in RNase free water to a final concentration of 10 ng × μL⁻¹. The analysis of the mitochondrial cytochrome b and the nuclear pyruvate kinase genes purchased from Microsynth (Balgach, Switzerland) was performed using SYBR Green real-time PCR (Roche Diagnostics, Rotkreuz, Switzerland) on an ABI PRISM 7700 sequence detector (PE Biosystems, Rotkreuz, Switzerland). Relative amounts of nuclear and mitochondrial DNA were determined by comparison of the amplified and quantified DNA from pyruvate kinase and cytochrome b (Primer sequences in Table 1). The amount of mitochondrial DNA was also normalized to citrate synthase activity.

Total RNA was extracted from Jurkat cells using RNeasy kit (Qiagen) with on column DNAse I digestion. Next 0.5 ug of RNA was used for M-MLV reverse transcription reactions (Invitrogen). For gRNA expression screening specific reverse primer (pX260-crRNA-3′/R, Table I.3 in S1 Experimental Procedures) was used in RT reaction followed by standard PCR using target A or B sense oligos as a forward primers (Table I.5 in S1 Experimental Procedures) and agarose gel electrophoresis. For checking neighboring genes expression oligo-dT primer mix was used in RT and cDNA was subjected to SybrGreen real time PCR (Roche) using mRNA specific primer pairs and β-actin as a reference (Table I in S1 Experimental Procedures).

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen, Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s protocol. One microgram of total RNA was reversed to cDNA using transcriptor first strand cDNA synthesis kit (Roche, Shanghai, China), followed by SYBR-Green real-time PCR (Roche, Shanghai, China). RT-PCR reactions were performed according to the manufacturer’s instructions. The 2 ^−ΔΔCt method was used to evaluate the mRNA expression. Relative expression was calculated and normalized to GAPDH. The sequences of oligonucleotide primers were synthesized by Sangon (Shanghai, China) and the forward and reverse primer sequences were as follows: GAPDH forward, 5′-ATCATCCCTGCCTCTACTGG-3′ and reverse, 5′-GTCAGGTCCACCACTGACAC-3′; AKR1C1 forward, 5′-CATGCCTGTCCTGGGATTT-3′ and reverse, 5′-AGAATCAATATGGCGGAAGC-3′.