A hematology analyzer (Siemens, Germany, ADVIA2120i), a PCR system (ABI Company, USA, 7500), a Total RNA Extraction Kit, an EasyPure miRNA Kit, a Reverse transcription+PCR Kit, and the TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China, ER601-01, AT351-01) were used. The miR-21 primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Table 1 ).
Transscript mirna first strand cdna synthesis supermix
Manufactured by Transgene
Sourced in China, United States
TransScript miRNA First-Strand cDNA Synthesis SuperMix is a laboratory product designed for the reverse transcription of miRNA into complementary DNA (cDNA). It provides a convenient and efficient method for the synthesis of first-strand cDNA from miRNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (14 mentions)
Easypure mirna kit, by Transgene (5 mentions)
Easyscript cdna synthesis supermix, by Transgene (4 mentions)
Transscript first strand cdna synthesis supermix, by Transgene (4 mentions)
Perfectstart green qpcr supermix, by Transgene (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Tb green premix ex taqtm 2, by Takara Bio (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Primer express, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Cfx96 real time qpcr detection system, by Bio-Rad (2 mentions)
Mirneasy serum plasma kit, by Qiagen (2 mentions)
Rnaiso plus reagent, by Takara Bio (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Total rna extraction kit, by Transgene (1 mentions)
Reverse transcription pcr kit, by Transgene (1 mentions)
Transzol up plus rna kit, by Transgene (1 mentions)
43 protocols using transscript mirna first strand cdna synthesis supermix
1
Quantifying miR-21 Expression
2
Quantitative Analysis of Defense Gene Expression in Radix pseudostellariae
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Plants were ground into powder with liquid nitrogen, and plant RNA was extracted with TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) in accordance with the instructions. Furthermore, RNA concentration was measured using NanoDrop 2000C Spectrophotometer (Thermo Scientific, United States). According to the kit’s instructions, the first-strand cDNA was synthesized using TransScript®, miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Each sample used 1 μg of total RNA, and the products were immediately diluted to 80 μl with DEPC water as a template.
Based on the previous transcriptome data of R. pseudostellariae in our laboratory (Qin et al., 2017 (link)), nine primer pairs were used (Supplementary Table 2 ) to analyze the expression of defense-related genes in R. pseudostellariae as a result of Trichoderma and/or F. oxysporum infection. The actin gene (Supplementary Table 2 ) was used as an internal reference gene. The 15 μl of the PCR reaction contains 7.5 μl of 2 × SYBR Green qPCR Master Mix (TransGen Biotech, Beijing, China), 1 μl of each primer (10 μM), 0.6 μl of cDNA template, and 5.9 μl H2O. The PCR program was as follows: 94°C for 30 s, followed by 40 cycles of 94 C for 5 s and 60°C for 30 s. After RT-PCR, the 2–ΔΔCT method (Livak and Schmittgen, 2001 (link)) was used to calculate the relative gene expression levels.
Based on the previous transcriptome data of R. pseudostellariae in our laboratory (Qin et al., 2017 (link)), nine primer pairs were used (
3
miRNA Extraction and Reverse Transcription
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MiRNA extraction was performed using the EasyPure® miRNA Kit, a miRNA extraction kit purchased from TransGen Biotech (Beijing, China). The reverse transcription reaction of the miRNA was performed using TransScript® miRNA First-Strand cDNA Synthesis SuperMix (containing tailing enzyme and reverse transcriptase), a reverse transcription kit purchased from TransGen Biotech (Beijing, China). The cDNA obtained from reverse transcription was diluted 5-fold for use in qRT-PCR.
4
Quantitative Analysis of Gene and miRNA Expression
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Total RNA from cells or tissues was extracted with Trizol reagent (Ambion, Life Technologies) and reverse transcribed into cDNA using the TransScript One‐Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing) or TransScript miRNA First‐Strand cDNA Synthesis SuperMix (TransGen Biotech) according to the manufacturers' instructions. The expression levels of different genes or miRNA were quantified by RT‐qPCR using the TransStartTop Green qPCR SuperMix Kit (TransGen Biotech). Real‐time quantitative PCR was performed by the Agilent StrataGene Mx3000P Multiplex Quantitative PCR System (Stratagene, Agilent). The results were analysed by StrataGene Mx3000P software with GAPDH (for mRNA) or U6 (for miRNA) as an internal control. The primers were listed in Table 1 .
5
Quantitative RT-PCR for miR-30 and Spastin mRNA
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TRIzol reagent (Invitrogen) was applied to total RNA extracts of HT22 or COS1 cells according to the manufacturer’s instructions. An EasyScript cDNA Synthesis SuperMix (TransGen Biotech, China) was used to reverse transcribe the mRNA. A TransScript® miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, China) was used for miRNA cDNA synthesis. The quantitative reverse transcription-polymerase chain reaction (PCR) was performed using TransStart Top Green quantitative PCR (qPCR) SuperMix (TransGen Biotech, China). miR-30 and mRNA expression levels were normalized to those of U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using a delta-delta-Ct method as described previously (Livak and Schmittgen, 2001 (link)). All PCR reactions were repeated at least three times. The primers specific to mature miR-30 were purchased from Qiagen. The following sense and anti-sense primers were used:
spastin (HT22): 5′-GAACCCGTCTTCTTTCTCGTC-3′, 5′-AATGGAGATGTACTCGAAGGC-3′;
spastin (COS1): 5′-AGCGGAACCTGTACTATTTCTC-3′, 5′-CTGCTTTCTCATCCTCATCGAT-3′;
MALAT1: 5′-CTCACTAAAGGCACCGAAGG-3′, 5′-GGCAGAGAAGTTGCTTGTGG-3′.
spastin (HT22): 5′-GAACCCGTCTTCTTTCTCGTC-3′, 5′-AATGGAGATGTACTCGAAGGC-3′;
spastin (COS1): 5′-AGCGGAACCTGTACTATTTCTC-3′, 5′-CTGCTTTCTCATCCTCATCGAT-3′;
MALAT1: 5′-CTCACTAAAGGCACCGAAGG-3′, 5′-GGCAGAGAAGTTGCTTGTGG-3′.
6
Gene Expression Profiling by RT-qPCR
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Total RNA was extracted with TRIzol (Beyotime, Beijing, China). The complementary DNA was synthesized using the EasyScript® First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China) and TransScript® miRNA First-Strand cDNA Synthesis SuperMix (Transgen) based on the instructions of the manufacturer. Afterwards, RT-PCR was carried out with the TransScript®Green Two-Step qRT-PCR SuperMix (Transgen) or TransScript® Green miRNA Two-Step qRT-PCR SuperMix (Transgen). The primer sequences were as follows: FOXD2-AS1-sence: 5′-TGGACCTAGCTGCAGCTCCA-3′, antisense: 5′-AGTTGAAGGTGCACACACTG-3′; miR-185-5p-sense: 5′-GCGGCGGTGGAGAGAAAGGCAG-3′, antisense: 5′-ATCCAGTGCAGGGTCCGAGG-3′; ROCK2-sence: 5′-AACGTCAGGATGCAGATGGG-3′, antisense: 5′-CAGCCAAAGAGTCCCGTTCA-3′; GAPDH-sence: 5′-GTTGCAACCGGGAAGGAAAT-3′, antisense: 5′-GCCCAATACGACCAAATCAGA-3′ and U6-sence: 5′-CAGCACATATACTAAAATTGGAACG-3′, antisence: 5′-ACGAATTTGCGTGTCATCC-3′.
7
Quantifying mRNA and miRNA in Cells and Exosomes
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The total RNA and miRNA in M0 macrophages and H1299 cells were extracted using the RNAsimple Total RNA Kit (DP419, TIANGEN, Beijing, China) and the miRcute miRNA Isolation Kit (DP501, TIANGEN), respectively. Total RNA in exosomes was extracted using the Exosomal RNA Isolation Kit (58000, NORGEN). RNA was quantified using Microvolume Spectrometer (Titertek Berthold, Germany). The PrimeScrptTM RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, Dalian, China) and TransScript miRNA First-Strand cDNA Synthesis SuperMix (AT351-01, TransGen Biotech, Beijing, China) were used for total RNA and miRNA reverse transcription, respectively. The TB Green Premix Ex TaqTM II (RR820A, TaKaRa, Dalian, China) and PerfectStart Green qPCR SuperMix (TG-AQ601-02, TransGen Biotech, Beijing, China) were used for amplification, respectively. The primers (Table 1 ) used in this study were synthesized by General Biosystems (Durham, NC, USA), and the expressions of mRNA and miRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6, respectively. The data were analyzed using the 2−ΔΔCt method (28 (link)).
8
Validating sRNA-seq Results by qRT-PCR
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We randomly selected seven DEMs to validate the sRNA‐seq results using quantitative real‐time PCR (qRT‐PCR). cDNA samples were synthesized from remanent total RNA from the Section 2.4 with TransScript® miRNA First‐Strand cDNA Synthesis SuperMix (TransGen Biotech). Furthermore, qRT‐PCR analysis was performed using TransScript® Green miRNA Two‐Step RT‐qPCR SuperMix (TransGen Biotech) on a CFX96 real‐time PCR Detection System (Bio‐Rad), and the following thermal cycling conditions were described as our previous study (Ren et al., 2023 (link)). The snRNA U6 was served as an internal reference gene, and other primers were designed by Primer Premier 5 (Table S9 ). All reactions were performed in triplicate, and the relative expression was calculated using the 2 − ΔΔ c t
9
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from the specimens and the cells using Trizol (Takara, Dalian, China) according to the manufacturer's instructions. Concentrations of total RNA were measured by reading the absorbance at OD260/280 nm.
RT-qPCR was carried out using the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). First-strand cDNAs for mRNA and miRNA were obtained using the Reverse Transcription Kit (Takara, Dalian, China) and TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) in accordance with the manufacturer's procedure, respectively. Quantitative PCR was performed using SYBR® Premix Ex Taq™ II (Takara) according to the manufacturer's instructions. GAPDH mRNA and endogenous U6 small nuclear RNA (snRNA) levels were assayed for normalization, respectively. 2-ΔΔCt method was used for relative quantification and the primer sequences used in the study were shown in Table2 .
RT-qPCR was carried out using the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). First-strand cDNAs for mRNA and miRNA were obtained using the Reverse Transcription Kit (Takara, Dalian, China) and TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) in accordance with the manufacturer's procedure, respectively. Quantitative PCR was performed using SYBR® Premix Ex Taq™ II (Takara) according to the manufacturer's instructions. GAPDH mRNA and endogenous U6 small nuclear RNA (snRNA) levels were assayed for normalization, respectively. 2-ΔΔCt method was used for relative quantification and the primer sequences used in the study were shown in Table
10
RNA Immunoprecipitation Assay for hnRNPA2B1
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The RIP assay was performed with a BersinBio RIP Kit (BersinBio, Guangzhou, China) following the manufacturer’s instructions. Briefly, Flag-tagged full-length and partial hnRNPA2B1 plasmids were synthesized by Comate Bioscience Co., Ltd. (Changchun, China). The cells were transfected with plasmids, lysed, and then incubated with an anti-Flag antibody (Sigma-Aldrich, F1804) overnight at 4 °C. Magnetic beads were then added to the lysate and, after 2 h of incubation, the RNA was purified and reverse transcribed using TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The levels of miR-6881-3p, miR-6726-3p, miR-34c-3p, and miR-4457 were analyzed using qPCR with IgG serving as a negative control.
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