FACS-sorted tumor RNA samples were treated using TRIzol (Ambion, Foster City, CA, USA), treated with ezDNase (Invitrogen), extracted using an RNA extraction kit (New England Biolabs, Ipswich, MA, USA), and reverse transcribed into cDNA using reverse transcriptase (Wisent Inc, Saint-Jean-Baptiste, QC Canada). Real-time quantitative PCR (qRT-PCR) reactions were performed on an CFX384 (Biorad, Hercules, CA, USA) in 384-well plates or 96-well iCycler (Biorad) containing 5–20 ng cDNA, 150 nM of each primer, and 5–10 μl 2X qPCR Master Mix (Wisent Inc) in a 10–20 μl total volume depending on the type of machine used. Primers were designed to span exon junctions using Primer3Plus, and were validated. Relative mRNA levels were calculated using the comparative Ct method normalized to Ppib and Hprt1 mRNA. The following primer sequences were used (Table 1 ):
Rna extraction kit
Manufactured by New England Biolabs
Sourced in United States
The RNA extraction kit is a laboratory tool designed to efficiently isolate and purify ribonucleic acid (RNA) from various biological samples. It provides a straightforward, reliable, and reproducible method for extracting high-quality RNA for downstream applications such as gene expression analysis, reverse transcription, and other molecular biology procedures.
Automatically generated - may contain errors
4 protocols using rna extraction kit
1
RNA Extraction and qRT-PCR Quantification
2
Morphine-Induced Regulation of OPRM1 Isoforms
3
Profiling Gene Expression in Stem Cells
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Total RNA was extracted from HDFa, iPSCs and hCOs using an RNA extraction kit (New England Biolabs) according to the manufacturer's instructions. RT-PCR reactions were performed as previously described (Donadoni et al. 2019) . Briefly, cDNAs were synthesized by using M-MLV Reverse Transcriptase (Invitrogen) followed by removal of RNA templates by RNase H digestion. A total of 50 ng cDNA was used as template for PCR reactions. Pluripotency markers OCT4 and SOX2, and differentiation markers, such as astrocytic structural marker, GFAP, neuronal differentiation markers, TUJ1 and MAP2, and oligodendroglia marker, Olig2, were amplified by PCR. GAPDH mRNA was amplified from the same set as internal controls. List of primers used is available in Table 2. Amplified products were resolved on 1% DNA agarose gels and visualized by ethidium bromide staining.
4
Ethanol-Induced Isoform Shifts in Mcl-1
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hNSPs, hNPCs, immature neurons, and mature neurons were exposed to 50 mM EtOH. At 6 and 24 h post-exposure time points, cells were harvested, and total RNA was extracted with an RNA extraction kit (New England Biolabs) according to the manufacturer's instructions. RT-PCR reactions were performed as described previously24 (link). After treatment with DNase I, followed by phenol/chloroform extraction and EtOH precipitation, cDNAs were synthesized from total RNA using M-MuLV reverse transcriptase. RNA templates were removed by RNase H digestion. A total of 100 ng cDNA was used as template for PCR reactions. Alternatively spliced isoforms of Mcl-1 were amplified by PCR using Mcl-1F: 5′- GGACACAAAGCCAATGGGCAGGT-3′ and Mcl-1R: 5′-GCAAAAGCCAGCAGCACATTCCTGA-3′. β-Actin mRNA was also amplified from the same set of RNA samples by RT-PCR as an internal control using F: 5′-CTACAATGAGCTGCGTGTGGC-3′ and R: 5′-CAGGTCCAGACGCAGGATGGC-3′. Amplified gene products were resolved on a 1.5% DNA-agarose gel.
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