Rt2 mirna first strand kit

Total RNA was first isolated from pelleted cells using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and miRNA fraction was purified using RT² qPCR-Grade miRNA isolation kit (SABiosciences, a Qiagen Company) in the first experiment. In the following experiments, miRNA was isolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany) and enriched by RNeasy MinElute Cleanup Kit (Qiagen). Two hundred nanograms of miRNA was converted to cDNA using RT² miRNA First Strand Kit (SABiosciences). cDNA samples were mixed with RT² qPCR Master Mix (SABiosciences) and distributed in every well of a PCR array plate (Mouse miFinder RT² miRNA PCR array by SABiosciences) profiling the expression of 88 most abundantly expressed and best characterized miRNA sequences in the mouse genome. The array plate contained also four housekeeping assays for normalizing the qPCR array data as well as duplicate controls for reverse transcription reaction and for the efficiency of PCR reaction. Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used to determine Ct-values of each well. The fold changes in Ct-values were calculated using the web-based data analysis program of SABiosciences.

Total of 166 ng small RNAs from 3 placentas at day 50 derived from every group of pregnancy (IVP, NT and AI) and donor cells (in triplicate) were synthesized into first strand cDNAs using RT² miRNA first strand kit (SABiosciences). Real time qPCR of miRNAs was performed using 384-well miRNAs primed PCR plate (SABiosciences) comprised of 377 individual miRNAs (most of them are conserved in human, mouse and bovine), 4 endogenous controls (U6, Snord44, Snord47 and Snord48), 2 reverse transcription controls and 2 positive PCR controls according to the protocols provided by the manufacturer. The assays were performed in ABI 7900 HT real time PCR system (Applied Biosystems, Foster City, CA, USA) with sybr green technology (SABiosciences). Data were analysed by ΔΔC_t method and normalization was performed by geometric mean of four endogenous controls through SAbiosciences’s PCR array data analysis on-line web-based analysis portal provided with t test. A fold regulation 2 or more with the value of P less than/equal to 0.05 were considered as significant different expression.

Total RNA samples obtained from MSCs under normoxia or hypoxia were sent to LC Sciences (Houston, TX) for miRNA microarray profiling. Data was analyzed by LC Sciences with in-house developed computer programs. Intensity values were transformed into log2 scale, and fold changes were given in log2 scale. A t-test was performed between normoxic MSCs and hypoxic MSCs, and statistical significance was considered at P<0.01. The microarray data were confirmed using an miRNA detection protocol with RT² miRNA First Strand Kit (SA biosciences). Computational miRNA target prediction analysis was performed with TargetScan (version 6.2) and miRDB to predict potential binding between VEGF 3′UTR and miRNA.

miRNA expression profiling was performed using the RT²miRNA PCR Array System (Qiagen, Inc. USA, Germantown, MD, USA) on the MyiQ Single-Color Real-Time PCR platform (Bio-Rad, Hercules, CA, USA). Briefly, 1.0 × 10⁶ cells were grown in 1.2 ml Matrigel in 30 mm-plates for 10 days (for T4-2 Rev, 350 nM AG1478 was added). The medium was removed and cells were scraped off from the dish with 2 ml phosphate-buffered saline (PBS) with 5 mM EDTA. Cells were spun down to harvest pellets, which were repetitively washed with ice-cold PBS + EDTA until the Matrigel was dissolved. The total RNA was extracted with 1 ml Trizol (Life Technologies) and purified with an RNeasy plus mini kit (Qiagen, Inc, USA) according to the manufacturers’ protocols. cDNA was generated from 4 μg of RNA using the RT²miRNA First Strand Kit (SABiosciences), mixed with SYBR Green Master Mix (SABioseicences) and loaded onto an array with 98 wells. Real-time PCR was performed according to the manufacturer’s instructions, and data analysis was performed using the manufacturer’s PCR Array Data Analysis Web Portal (Qiagen, Inc, USA).

As we described previously 21 (link), the extracted total RNA including miRNAs (10 ng/µl concentration) was first reverse transcribed into first strand cDNA using the RT2- miRNA First Strand Kit following manufacturer's recommendations (SA Biosciences, Rockville, MD). One µl cDNA per well was then mixed with SYBR Green qPCR Master Mix and placed into a 96-well PCR-array plate containing a panel of 88 mature miRNAs sequences. The arrays also contain appropriate small nucleolar RNA sequences that are used as housekeeping assays and quality controls. One µl was used in a 12 µl final volume reaction for Real-time PCR analysis on an Applied Biosystems Step-One Plus Real Time PCR system. Relative amounts were calculated by the ΔΔ CT method. Samples without good RNA quality were excluded in the statistical analysis.