Total RNA was extracted from mouse testis following the manufacturer's protocol using the TRI Reagent (Sigma, T9424). Total RNA was reverse transcribed into cDNA by Hifair 1st Strand cDNA Synthesis Super Mix for qPCR (gDNA digester plus) (11141ES10, YEASEN, Shanghai, China). Slc25a26 gene was cloned from cDNA using nested PCR by Prime-STAR HS DNA Polymerase (R010A, TaKaRa) with 2 primer pairs (the first pair: forward, 5′-AAGTAGTTGCTCCATATCCCG-3′; reverse, 5′-TTGTAGCCAGTTTGCTTTCC-3′; the 2nd pair: forward, 5′-CTAGCTAGCCACCATGGACGCGCCGGGCT-3′; reverse, 5′-CTGCTAA CCGGTGGTGGGCTCTTCCTGCCCACC-3′). PCR products were digested with NheI and AgeI (NEB Inc., MA, USA) and ligated into PVAX1 vector with FLAG tag. cRNAs were in vitro transcribed and capped from linearized plasmids using mMESSAGE mMACHINE Kit (AM1344, Invitrogen) and Poly(A) Tailing Kit (AM1350, Invitrogen) according to the manufacturer's instruction. Synthesized RNA was aliquoted and stored at -80°C.
Hifair 1st strand cdna synthesis super mix for qpcr gdna digester plus
Manufactured by Yeasen
Sourced in China
Hifair 1st Strand cDNA Synthesis Super Mix for qPCR (gDNA digester plus) is a reagent kit designed for the reverse transcription of RNA into cDNA. The kit includes a gDNA digester component to remove genomic DNA contamination. The cDNA synthesis is optimized for real-time PCR (qPCR) applications.
Automatically generated - may contain errors
Lab products found in correlation
Tri reagent, by Merck Group (2 mentions)
Poly a tailing kit, by Thermo Fisher Scientific (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Mmessage mmachine kit, by Thermo Fisher Scientific (1 mentions)
Hieff qpcr sybr green master mix high rox plus, by Yeasen (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Yeasen (1 mentions)
Goat anti rabbit igg h l hrp fms rb01, by Fcmacs (1 mentions)
Mouse s0002, by Affinity Biosciences (1 mentions)
Remodelin, by CSNpharm (1 mentions)
Nat10, by Proteintech (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Puromycin, by Merck Group (1 mentions)
Sybr green pcr master mix, by Yeasen (1 mentions)
β actin 60008 1 ig, by Proteintech (1 mentions)
Blazetaq sybr green qpcr mix 2, by GeneCopoeia (1 mentions)
Taq pcr master mix, by GeneCopoeia (1 mentions)
4 protocols using hifair 1st strand cdna synthesis super mix for qpcr gdna digester plus
1
Mouse Testis RNA Extraction and cRNA Synthesis
2
Quantifying Gene Expression in Oocytes and Embryos
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A total of 25-30 oocytes/zygotes or 20 cleavage embryos or 15 blastocysts were collected, respectively. Total RNA was extracted using the TRI Reagent (Sigma, T9424) and reverse transcribed into cDNA by Hifair 1st Strand cDNA Synthesis Super Mix for qPCR (gDNA digester plus) (11141ES10, YEASEN, Shanghai, China). Each PCR reaction consisted of 10 μl of Hieff qPCR SYBR Green Master Mix (High Rox Plus) (11203ES03, YEASEN, Shanghai, China), 8.2 μl of water, 1 μl of cDNA sample, and 0.8 μl of gene-specific primers (5 μM; Slc25a26 primers: forward, 5′-TCTGGGGCAACAGTGTGTAG-3′; reverse, 5′-TACTAAGTGTGTGCGGCGGT-3′; mt-Cytb primers: forward, 5′-ACCTCAAAGCAACGAAGCCT-3′; reverse, 5′-TGGGTGTTCTACTGGTTGGC-3′). Real-time PCR was performed by the StepOnePlus Real-Time PCR system (Applied Biosystems, USA). mRNA levels were normalized to Gapdh. The experiments were performed for at least three times.
3
Protein Expression Profiling in Cell Lines
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The primary antibodies used in this study were at the dilutions of 1:1000 as follows: NAT10 (13365-1-AP, Proteintech), ac4C (ab252215, Abcam), AKT (9272s, Cell Signaling Technology), p-AKT (4058s, Cell Signaling Technology), BCL-XL (2762s, Cell Signaling Technology), Bax (2774s, Cell Signaling Technology), PARP (9542S, Cell Signaling Technology), Cleaved Caspase-3 (9661S, Cell Signaling Technology), β-actin (60008-1-Ig, Proteintech), CDK4 (11026-1-AP, Proteintech), CDK6 (14052-1-AP, Proteintech).
The second antibodies included goat anti-Rabbit IgG(H+L) HRP (FMS-Rb01, Fcmacs) or mouse (S0002, Affinity) were at the dilutions of 1:5000.
Remodelin was purchased from CSNpharm (Chicago, USA). Puromycin was obtained from Merck KGaA (Darmstadt, Germany). Trizol reagent (YEASEN, Shanghai), Hifair 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (YEASEN, Shanghai), SYBR Green PCR master mix (YEASEN, Shanghai).
The second antibodies included goat anti-Rabbit IgG(H+L) HRP (FMS-Rb01, Fcmacs) or mouse (S0002, Affinity) were at the dilutions of 1:5000.
Remodelin was purchased from CSNpharm (Chicago, USA). Puromycin was obtained from Merck KGaA (Darmstadt, Germany). Trizol reagent (YEASEN, Shanghai), Hifair 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (YEASEN, Shanghai), SYBR Green PCR master mix (YEASEN, Shanghai).
4
Isoform-specific RT-qPCR protocol for AS events
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To distinguish AS events, the reverse transcription (RT)-PCR was performed. Brie y, the RNA used for Iso-Seq was isolated using EZ-10 DNA away RNA Mini-Preps Kit (Sangon, https://www.sangon.com) and synthesized to complementary DNA using the Hifair ® 1st Strand cDNA Synthesis SuperMix for qPCR gDNA digester plus (Yeasen). Isoform-speci c primers were designed using Vector NTI software (Supplementary Table S1). PCR ampli cation was performed using 2×Taq PCR Master Mix (K1034, https://www.apexbt.com/) and the PCR products were showed in agarose gel stained with GoodView TM Nucleic Acid Stain (Apr-13-2021, HGV-, https://www.sbsbio.com).
BlazeTaq TM SYBR ® Green qPCR Mix 2.0 (GeneCopoeia, USA) was used for RT-qPCR reactions on a CFX96 Real-time System (BIO-RAD, USA). The ampli cation program was brie y described in previous study (Wagner 2013 (link)). To ensure the precise and reproducible results, each sample was replicated three times and Ef1α in potato were used for each sample as an endogenous control (Supplementary Table S1). The formula F = 2 -ΔΔCt was used to calculate the relative expression levels of selected genes.
BlazeTaq TM SYBR ® Green qPCR Mix 2.0 (GeneCopoeia, USA) was used for RT-qPCR reactions on a CFX96 Real-time System (BIO-RAD, USA). The ampli cation program was brie y described in previous study (Wagner 2013 (link)). To ensure the precise and reproducible results, each sample was replicated three times and Ef1α in potato were used for each sample as an endogenous control (Supplementary Table S1). The formula F = 2 -ΔΔCt was used to calculate the relative expression levels of selected genes.
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