RNA was isolated from cells using the GENEzol TriRNA Pure Kit (GeneAid). cDNA synthesis was carried out with the Quanta cDNA Reverse Transcription Kit (QuantaBio). qPCR was performed with the iTaq Supermix (BioRad) on the BioRad iCycler. For BCR-ABL quantification, mononuclear cells derived from peripheral blood of CML patients were isolated on Ficoll-Hypaque gradient and total RNA was isolated. Real-time for BCR-ABL quantification was performed using ipsogen® BCR-ABL1 Mbcr IS-MMR (Qiagen). The comparative Ct method was employed to quantify transcripts, and delta Ct was measured in triplicate. Primers used in this study are provided in Supplementary Table S3 .
Quanta cdna reverse transcription kit
Manufactured by Quantabio
Sourced in United States
The Quanta cDNA Reverse Transcription Kit is a laboratory tool designed for the conversion of RNA to complementary DNA (cDNA). It provides the necessary components, including reverse transcriptase enzyme, reaction buffer, and primers, to facilitate this fundamental step in gene expression analysis and other molecular biology applications.
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5 protocols using quanta cdna reverse transcription kit
1
BCR-ABL Quantification in CML
2
RNA Quantification Using qPCR
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RNA was isolated from cells using the GENEzol TriRNA Pure Kit (GeneAid). cDNA synthesis was carried out with the Quanta cDNA Reverse Transcription Kit (QuantaBio). Then, qPCR was performed with the iTaq Supermix (BioRad, Hercules, CA, USA) on the Biorad iCycler. The comparative Ct method was employed to quantify transcripts, and delta Ct was measured in triplicate.
Sequences of used primers were the following: Cyclophilin A (Fw: GTCAACCCCACCGTGTTCTT Rv: CTGCTGTCTTTGGGACCTTGT), U2AF65 (Fw: 5′ACCCAGGCTATGGCCTTTG, Rv: 5′GAAGCGGCTGGTAGTCGTG), Jmjd6 total (Fw: 5′GGGTGCGTTAGTGTCAGGAA, Rv: 5′CCTTTCCACGTTATCCGCCA), Jmjd6 Exon 5 Inclusion (Fw: 5′ACCTGGAGGGACCAGCTC, Rv: 5′TCTGAGTCGGAGTCTGACGA), Jmjd6 Exon 5 Exclusion (Fw: 5′CAAGGAAATGGTATAGGATTTTGAA, Rv: 5′TTTGCTGACACAGTCGTCCT).
Sequences of used primers were the following: Cyclophilin A (Fw: GTCAACCCCACCGTGTTCTT Rv: CTGCTGTCTTTGGGACCTTGT), U2AF65 (Fw: 5′ACCCAGGCTATGGCCTTTG, Rv: 5′GAAGCGGCTGGTAGTCGTG), Jmjd6 total (Fw: 5′GGGTGCGTTAGTGTCAGGAA, Rv: 5′CCTTTCCACGTTATCCGCCA), Jmjd6 Exon 5 Inclusion (Fw: 5′ACCTGGAGGGACCAGCTC, Rv: 5′TCTGAGTCGGAGTCTGACGA), Jmjd6 Exon 5 Exclusion (Fw: 5′CAAGGAAATGGTATAGGATTTTGAA, Rv: 5′TTTGCTGACACAGTCGTCCT).
3
Quantifying SAT1 Splice Variant Expression
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RNA was isolated from cells using the GENEzol TriRNA Pure Kit (GeneAid). cDNA synthesis was carried out with the Quanta cDNA Reverse Transcription Kit (Quantabio). Then, qPCR was performed with the iTaq Supermix (Bio-Rad) on the Bio-Rad iCycler. The comparative Ct method (ΔΔCT) was used to quantify transcripts, and delta Ct was measured in triplicate. The amount of target, normalized to the CycloA reference gene for total mRNA and normalized to total mRNA for the splice variant, is given by the arithmetic formula: 2–ΔΔCT, where ΔΔCT = ΔCT test sample–ΔCT reference sample. PSI was calculated by the ratio of SAT1 exon X and SAT1 total mRNA amount. Primers used to amplify the target genes are provided in Supplemental Table S1 .
4
Sensitive qPCR Quantification of Gene Expression
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RNA was isolated from cells using the GENEzol TriRNA Pure Kit (GeneAid). cDNA synthesis was carried out with the Quanta cDNA Reverse Transcription Kit (QuantaBio). qPCR was performed with the iTaq Supermix (BioRad) on the Biorad iCycler. The comparative Ct method was used to quantify transcripts, and delta Ct was measured in triplicate. Primers used in this study are provided in Supplemental Table S1 .
5
RNA Extraction and qRT-PCR from Saliva
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Saliva was kept at −20 °C before extraction. Saliva was centrifuged at 4 °C for 15 min at 2600 g. RNA was extracted from 400 μL of the supernatant using the NucleoSpin RNA XS Kit (Macherey-Nagel, Dueren, Germany) according to the manufacturer’s instructions, with elution in 30 μL. cDNA synthesis was carried out with 23 μL of RNA using the Quanta cDNA Reverse Transcription Kit (QuantaBio, Beverly, MA, USA) according to the manufacturer’s instructions. Real-time PCR was performed with the iTaq Supermix (BioRad Rishon Le Zion, Israel) on the Bio-Rad iCycler. The comparative Ct method was used to quantify transcripts, and ∆Ct was measured in triplicate. The primers used in this study are provided in Supplementary Table S1 .
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