For miRNA qRT-PCR the miScript SYBR® Green PCR Kit (Qiagen) and for mRNA the SYBR® Green PCR Master Mix (Applied Biosystems®) was used. In both instances, the manufacturer’s instructions were followed. Each cDNA sample was tested in duplicate (as in comparable studies24 (link)–26 (link) and dCT was determined by normalization of the Ct value to the Ct value of the reference small nucleolar RNA SNORD61 (Qiagen) for miRNA or to beta-actin (Invitrogen, Carlsbad, USA) for mRNA. Data were transformed into relative values by calculating: 2−ΔΔCT (ddCT) as previously described23 (link).
Snord61
Manufactured by Qiagen
Sourced in United States
SNORD61 is a small nucleolar RNA (snoRNA) that is involved in the modification of other cellular RNAs. snoRNAs are a class of small RNA molecules that guide the chemical modification of other RNAs such as ribosomal RNAs, small nuclear RNAs, and transfer RNAs.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy mini kit, by Qiagen (3 mentions)
Miscript 2 rt kit, by Qiagen (3 mentions)
Miscript sybr green pcr kit, by Qiagen (2 mentions)
Snord68, by Qiagen (2 mentions)
Miscript universal primer, by Qiagen (2 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Nanophotometer, by Implen (1 mentions)
Rt2 profiler pcr array, by Qiagen (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Qscript one step sybr green qrt pcr kit, by Quanta Biosciences (1 mentions)
Qscript microrna cdna synthesis kit, by Quanta Biosciences (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
5 protocols using snord61
1
qRT-PCR for miRNA and mRNA Quantification
2
Quantitative Analysis of miRNA Expression
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Total RNA containing microRNA was extracted using a miRNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. The quantity of isolated RNA was measured using a NanoPhotometer (IMPLEN, München, Germany), and 1 μg of RNA was reverse-transcribed using a miScript II RT kit (QIAGEN). The miScript universal primer (QIAGEN) was used as the antisense primer. SNORD61 and SNORD68 (QIAGEN) were used as the internal controls. The following primers were used: hsa-let-7a-5p TGA GGT AGT AGG TTG TAT AGT T, hsa-let-7b-5p TGA GGT AGT AGG TTG TGT GGT T, hsa-let-7c-5p TGA GGT AGT AGG TTG TAT GGT T, hsa-let-7d-5p AGA GGT AGT AGG TTG CAT AGT T, hsa-let-7e-5p TGA GGT AGG AGG TTG TAT AGT T, hsa-let-7f-5p TGA GGT AGT AGA TTG TAT AGT T, hsa-let-7g-5p TGA GGT AGT AGT TTG TAC AGT T and hsa-let-7i-5p TGA GGT AGT AGT TTG TGC TGT T.
3
miRNA and Cancer Stem Cell Expression Analysis
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qPCR was performed as described previously.47 (link) For the miRNA PCR arrays, we purchased the miScript miRNA PCR Array kit (Human Cancer PathwayFinder) from QIAGEN (Valencia, CA, USA). Total RNA containing miRNA was extracted using an miRNeasy Mini Kit (QIAGEN), according to the manufacturer's protocol. To confirm let-7 miRNA expression, 1 μg of small RNA was reverse-transcribed using a miScript II RT kit (QIAGEN). The miScript universal primer (QIAGEN) was used as the antisense primer. SNORD61 and SNORD68 (QIAGEN) were used as the internal controls. The primers used for qPCR are listed in the Supplementary Table S4 . For the cancer stem cell arrays, we used RT2 Profiler PCR Arrays (QIAGEN).
4
qRT-PCR Analysis of Ezh2, Gapdh, GFP
5
Quantification of miRNA-128 Expression
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Total RNA, including miRNA, was extracted from cells using the miRNeasy Mini Kit (QIAGEN, Mississauga, ON, Canada). cDNA for gene expression was synthesized using oligo-dT and reverse transcriptase (Thermo Fisher Scientific). cDNA of miRNA-128 was synthesized using the qScript microRNA cDNA synthesis kit (Quanta Biosciences, San Francisco, CA, USA) or miScript II RT Kit (QIAGEN) according to the manufacturers’ instructions. The cDNA was then subjected to quantitative PCR (qPCR) with respect to miRNA-128 expression using the qScript One-Step SYBR Green qRT-PCR kit (Quanta Biosciences) or miScript SYBR Green PCR Kit (QIAGEN). qPCR was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada). SNORD61 (QIAGEN) was used as an internal loading reference. PCR cycling conditions for qScript One-Step SYBR Green qRT-PCR kit were as follows: 95°C for 2 min followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s, or 95°C for 15 min followed by 40 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 30 s for the miScript SYBR Green PCR Kit.
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