Total RNA was purified from control HeLa cells and HeLa cells constitutively expressing HSUR2 stored in TRIzol® according to the manufacturer's instructions (Ambion). RNA was suspended in 85 μl of water, 10 μl of 10× DNAse reaction buffer (New England Biolabs), and 10 units (5 μl) of RNAse-free DNAse I (New England Biolabs) and incubated at 37°C for 30 min. cDNA was synthesized in 20 μl reactions from 0.9 μg of DNase I-treated total RNA using the High-Capacity cDNA Reverse Transcription Kit with MultiScribe Reverse Transcriptase and random primers (Applied Biosystems). Real-time PCR was performed in 8 μl reactions using primers at 0.5 μM and KAPA SYBR green master mix (KAPA Biosystems) in a Roche 480 Light Cycler as previously described (17 (link),18 (link)). All reactions were performed in triplicate in each independent experiment. Relative expression of HSUR2 target mRNAs was calculated as previously described (17 (link)).
Multiscribe reverse transcriptase and random primers
Manufactured by Thermo Fisher Scientific
Sourced in United States
MultiScribe Reverse Transcriptase is an enzyme used in the conversion of RNA to complementary DNA (cDNA). It works in conjunction with random primers to initiate the reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Multiscribe mirna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Kapa sybr green master mix, by Roche (1 mentions)
Rnase free dnase 1, by New England Biolabs (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Universal rna purification kit, by EURx (1 mentions)
Rnase free dnase 1, by EURx (1 mentions)
Simplysafe, by EURx (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Mirna assay, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
7500 fast real time pcr system, by Bio-Rad (1 mentions)
6 protocols using multiscribe reverse transcriptase and random primers
1
Quantitative RT-PCR Analysis of HSUR2 Targets
2
Extraction and Reverse Transcription of Total RNA
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Total RNA was extracted from liquid nitrogen‐frozen powders of leaves and roots of uninfected and infected plants using the universal RNA purification kit (EURx, Gdańsk, Poland) according to the manufacturer’s instructions. During extraction, samples were treated with RNase‐free DNase I (EURx) directly on columns to digest contaminating genomic DNA. The RNA amount was measured spectrophotometrically (NanoDrop ND‐1000; Thermo Scientific, Waltham, MA, USA), and its purity and integrity were verified on 1.2% (w/v) agarose gel containing SimplySafe (EURx). Horizontal electrophoresis was run in 1 × TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). Purified total RNA samples (2 μg) were reverse‐transcribed using a high‐capacity cDNA reverse transcription kit with MultiScribe Reverse Transcriptase and random primers (Applied Biosystems/Thermo Scientific).
3
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from whole placental halves using the RNeasy Plus Mini Kit (Qiagen, UK) whereas total RNA was extracted from TS and ES cells using trizol (Invitrogen), chloroform and ethanol extraction. Multiscribe Reverse Transcriptase and random primers (Applied Biosystems) were used to synthesize cDNA from 2.5 ug of RNA. Samples were analysed in duplicate by qRT-PCR (7500 Fast Real-Time PCR System, Applied Biosystems, UK) using Sybr green chemistry and pairs of forward and reverse primers (Figure 7—source data 2 ). The expression of genes of interest were normalized to the expression of Gapdh or Sdha which was not affected by genotype. Data were analysed using the 2–ΔΔCT method for quantification.
4
Quantifying miRNA and mRNA Levels
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Total RNA was extracted using the miRNEasy kit (Qiagen). Reverse transcription was performed using either Multiscribe reverse transcriptase and random primers (Applied Biosystems, Foster City, CA, USA) to generate cDNA, or the Multiscribe miRNA Reverse Transcription kit (Applied Biosystems) using miRNA-specific primers to produce miRNA. Quantitative PCR was performed using inventoried miRNA assays (Applied Biosystems) with standard ABI protocols and reagents, as previously described [19 (link)]. The fold changes between groups were evaluated using relative quantization (delta Ct method) with b-actin as an endogenous mRNA control and RNU48 as an endogenous miRNA control. All the qPCR analyses were repeated at least three times to confirm differences in the expression levels and only results consistent across all three analyses were considered valid. For the mouse striatum, gene expression of miR-132 was measured. For the mouse cortex, expressions of BAG1 [20 (link)] and SAG1 [21 (link)] were measured.
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from HeLa cells using the RNeasy Plus Mini Kit (TransGen, China). Multiscribe Reverse Transcriptase and random primers (Applied Biosystems) were used to synthesize cDNA from 1 ug of RNA. Samples were analyzed in duplicate by qRT-PCR (7500 Fast Real-Time PCR System, Bio-Rad, USA) using Sybr green chemistry and pairs of forward and reverse primers. The expression of genes of interest was normalized to the expression of GAPDH which was not affected by genotype. Data were analyzed using the 2-ΔΔCT method for quantification.
6
Quantification of miRNA and mRNA Levels
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