Total RNA was extracted using an RNA extraction kit (Tiangen Biotech, Beijing, China). For mRNA detection, the SuperScript IV reverse transcriptase system (Thermo Fisher Scientific, Carlsbad, CA, USA) was used for cDNA synthesis. PCR reactions were performed using the SYBR® Premix Ex Taq™ (Takara, Dalian, China). For miRNA detection, RNA was reverse transcribed into cDNA by using the miRNA First-Strand cDNA synthesis kit (Tiangen). PCR was conducted using the miRNA qPCR detection kit (Tiangen). Data were processed with the 2−ΔΔCt method after normalizing to GAPDH or U6. Primer sequences are given in Additional file 1 : Table S2.
Superscript 4 reverse transcriptase system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The SuperScript® IV Reverse Transcriptase system is a reverse transcriptase enzyme used for the conversion of RNA into complementary DNA (cDNA). It is designed for high-sensitivity and high-fidelity cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Qrt pcr primers of h3, by Qiagen (3 mentions)
H3 3a gene, by Qiagen (3 mentions)
Mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Rna extraction kit, by Tiangen Biotech (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Mirna qpcr detection kit, by Tiangen Biotech (1 mentions)
Thermopol buffer, by New England Biolabs (1 mentions)
5 protocols using superscript 4 reverse transcriptase system
1
Comprehensive RNA Extraction and Analysis
2
Quantitative Analysis of H3.3 Gene Expression
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Total mRNAs were isolated from cells using RNeasy mini kit (Qiagen, Hilden, Germany). Complementary DNAs were produced using the SuperScript® IV Reverse Transcriptase system (Thermo Fisher Scientific). qRT-PCR was carried out using Power SYBR Green master mix (Thermo Fisher Scientific). The qRT-PCR primers of H3.3 (i.e., H3-3A gene) was obtained from Qiagen (Cat. No. QT00247128). Gene expression levels were normalized to GAPDH using primers (forward: 5ʹ-GGAAGGTGAAGGTCGGAGTCA-3ʹ and reverse: 5ʹ-GTCATTGATGGCAACAATATCCACT-3ʹ).
3
Reverse Transcription and PCR Protocol
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SuperScript IV reverse transcriptase system (Thermo Fisher Scientific, Carlsbad, CA, USA) was used to perform reverse transcription. Polymerase chain reaction (PCR) was performed using Taq DNA polymerase with ThermoPol buffer (New England BioLabs, Ipswich, MA, USA), with initial denaturation at 95 °C for 30 s, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 68 °C for 10 s. Final extension was performed at 68 °C for 5 min. Electrophoresis was performed using 1.5% agarose gel.
4
Quantifying H3.3 Gene Expression
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Total mRNAs were isolated from cells using RNeasy mini kit (Qiagen, Hilden, Germany).
Complementary DNAs were produced using the SuperScript® IV Reverse Transcriptase system (Thermo Fisher Scientific). qRT-PCR was carried out using Power SYBR Green master mix (Thermo Fisher Scientific). The qRT-PCR primers of H3.3 (i.e. H3-3A gene) was obtained from Qiagen (Cat. No. QT00247128). Gene expression levels were normalized to GAPDH using primers (forward: 5'-GGAAGGTGAAGGTCGGAGTCA-3' and reverse: 5'-GTCATTGATGGCAACAATATCCACT-3').
Complementary DNAs were produced using the SuperScript® IV Reverse Transcriptase system (Thermo Fisher Scientific). qRT-PCR was carried out using Power SYBR Green master mix (Thermo Fisher Scientific). The qRT-PCR primers of H3.3 (i.e. H3-3A gene) was obtained from Qiagen (Cat. No. QT00247128). Gene expression levels were normalized to GAPDH using primers (forward: 5'-GGAAGGTGAAGGTCGGAGTCA-3' and reverse: 5'-GTCATTGATGGCAACAATATCCACT-3').
5
Quantifying H3.3 Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mRNAs were isolated from cells using RNeasy mini kit (Qiagen, Hilden, Germany).
Complementary DNAs were produced using the SuperScript® IV Reverse Transcriptase system (Thermo Fisher Scientific). qRT-PCR was carried out using Power SYBR Green master mix (Thermo Fisher Scientific). The qRT-PCR primers of H3.3 (i.e. H3-3A gene) was obtained from Qiagen (Cat. No. QT00247128). Gene expression levels were normalized to GAPDH using primers (forward: 5'-GGAAGGTGAAGGTCGGAGTCA-3' and reverse: 5'-GTCATTGATGGCAACAATATCCACT-3').
Complementary DNAs were produced using the SuperScript® IV Reverse Transcriptase system (Thermo Fisher Scientific). qRT-PCR was carried out using Power SYBR Green master mix (Thermo Fisher Scientific). The qRT-PCR primers of H3.3 (i.e. H3-3A gene) was obtained from Qiagen (Cat. No. QT00247128). Gene expression levels were normalized to GAPDH using primers (forward: 5'-GGAAGGTGAAGGTCGGAGTCA-3' and reverse: 5'-GTCATTGATGGCAACAATATCCACT-3').
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