Rneasy midi extraction kit

The expression of MIF, CD74, CXCR2, CXCR4, GAPDH, GUSB, EEFLA1 and TBP mRNAs was evaluated by quantitative RT-PCR analysis. Total mRNAs from each cell lines was extracted by Qiagen RNEasy Midi extraction Kit (Qiagen) according to manufacturer's instructions.
Reverse transcription was performed from 0.5 μg of total RNA using PrimerScript reverse transcriptase enzyme (Takara bio inc. Kit) and a mix of random hexamers – oligo d(T) primers, following suppliers instructions.
Real-time RT-PCR was performed on each sample in triplicate performed from 500 ng cDNA using an ABI 7900HT SDS system with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, California). SYBR green primers were designed using the program Primer Express v 2.0 (Applied Biosystems) with default parameters and obtained from Invitrogen. Primer sequences for the targeted human genes are available upon request. Results were normalized to the expression levels of GAPDH, GUSB, EEFLA1 and TBP expression genes, used as housekeeping genes. Normalisation factor and fold changes were calculated using the GeNorm method [43 (link)].

The lower lobe of the right lung was chopped into small pieces and immediately snap-frozen in liquid nitrogen. Twenty milligrams of lung tissue was homogenized and total RNA isolated (RNeasy Midi Extraction Kit, Qiagen, Australia) and reserve-transcribed into cDNA (SuperScript III reverse transcriptase; Invitrogen). Genes of interest were measured by qPCR/Taqman. Five microliters of cDNA from each sample was plated onto a 96-well PCR plate and submitted to the MHTP Genomics Facility, Hudson Institute of Medical Research. Quality control testing was performed using Sybr chemistry for the housekeeping gene 18s (ABI 7900 HT qPCR). This was followed by preamplification, then taqman analysis of the seven genes listed in Table 1. Relative mRNA levels of gene expression are expressed relative to the mean of the UNOP group.

For selected genes, gene expression and stability were monitored by RT-qPCR. For expression analyses, a volume equivalent to 2 mg of cell dry weight was harvested by centrifugation (1 min, 13,000 × g) and flash-frozen in liquid nitrogen. For stability analyses, the same procedure was applied and sampling was performed at times 0, 0.33, 1, 2, 5, 7, 9, and 13 min after rifampin addition (500 μg·ml⁻¹) for exponential-phase analysis and at times 0, 1, 3, 5, 7, 9, 13, and 17 min after rifampin addition for the glucose exhaustion phase. Extractions of total RNA were performed using a Qiagen RNeasy Midi extraction kit. Samples were treated with DNase to eliminate genomic DNA; the samples were subjected to reverse transcription using Super Script II reverse transcriptase (Life Technology). RT-qPCR was performed using a SYBR green-based detection protocol (Life Technology) with an ICycler real-time PCR detection system (Bio-Rad) and MyIQ software (Bio-Rad). Primers (Table 2) were designed with primer3 online freeware (http://frodo.wi.mit.edu/). t_1/2 values were determined as the inverse of the slope of the threshold cycle (C_T) over time.