RNA was extracted from the collected peach fruit tissues using a Total RNA Kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. RNA samples were evaluated in 1% agarose gels to determine the levels of degradation and contamination and then analyzed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, USA) to determine RNA quantity and quality. First-strand cDNA was synthesized from the RNA samples with a Reverse Transcriptase kit (Tiangen, Beijing, China). The cDNA products were diluted to 20 ng µL−1 for subsequent quantitative reverse-transcription PCR (qRT-PCR) analyses.
Reverse transcriptase kit
Manufactured by Tiangen Biotech
Sourced in China
The Reverse Transcriptase Kit is a laboratory tool used to convert RNA into complementary DNA (cDNA) molecules. This kit contains the necessary reagents and enzymes to perform the reverse transcription process, a crucial step in various molecular biology techniques such as gene expression analysis and RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr green, by Tiangen Biotech (2 mentions)
Total rna isolation kit, by Tiangen Biotech (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Total rna kit, by Sangon (1 mentions)
Trizol reagent kit, by Tiangen Biotech (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Rna extraction kit, by Tiangen Biotech (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Sybr green real time pcr master mix, by Bio-Rad (1 mentions)
Rnaprep pure cell bacteria kit, by Tiangen Biotech (1 mentions)
Mirneasy serum plasma kit, by Tiangen Biotech (1 mentions)
Primer sequence, by Sangon (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Minibest universal genomic dna extraction kit ver 5, by Takara Bio (1 mentions)
Reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Rnaprep pure plant plus kit, by Tiangen Biotech (1 mentions)
Sybr green master mix, by Tiangen Biotech (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rna isolation kit, by Tiangen Biotech (1 mentions)
18 protocols using reverse transcriptase kit
1
Total RNA Extraction and RT-qPCR Analysis
2
Quantification of mRNA Levels in Mouse Liver
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Total RNA was extracted from mouse liver tissues or cells with TRIzol Reagent Kit (Lot: DP431, Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the concentration and purity of RNA were examined using a spectrophotometer. cDNA was synthesized from 1 mg of total RNA using a reverse transcriptase kit (Lot: KR106, Tiangen Biochemical Technology (Beijing) Co., Ltd.). Primer sequences outlined in Table 1
3
Quantitative real-time PCR analysis
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Total RNA was extracted using TRI Reagent (Sigma), then reverse transcription was performed using a reverse transcriptase kit (Tiangen). Complementary DNA was used as a template, and quantitative real-time PCR experiments were performed using the GoTaq qPCR Master Mix. β-actin was used as a normalization control. The primer sequences are provided in Supplementary information .
4
Comprehensive RNA Extraction and qRT-PCR Analysis
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The total RNA was extracted from duodenum and ileum tissues using an RNA Extraction Kit (Tiangen, China) following the manufacturer's instructions. The complementary DNA (cDNA) was synthesized from 2 μg of total RNA by reverse transcription in a 20 μl reaction mixture using a reverse transcriptase kit (Tiangen, China). The reverse transcription procedure was employed as follows: 42°C for 15 min and 95°C for 3 min. The cDNA samples were stored at −20°C. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out on a Bio-Rad CFX96 Touch Real-Time PCR Detection System using the SYBR Green Real-time PCR Master Mix. Target gene expression was quantified using the 2−ΔΔCt method. The real-time PCR procedure was employed as follows: denaturation at 95°C for 2 min, followed by 39 cycles at 95°C for 0.5 s and 60°C for 10 s. The primers are listed in Table 2 .
5
Validation of Microarray Data by qRT-PCR
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The microarray data were validated by qRT-PCR studies on an independent cohort of 26 T2DM patients with CLI and 30 T2DM patients without CLI using SYBR Green (TIANGEN, China; catalog number FP411) according to a previously described method [24 (link)]. RNA was reverse-transcribed to cDNA using a reverse transcriptase kit (TIANGEN; catalog number: KR211) according to the manufacturer's instructions. miRNAs were then analyzed by qRT-PCR using the following conditions: 95°C for 1 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 70°C for 10 s. Duplicate assays were performed for each sample. The hsa-miR-93-5p value from the duplicate was used as the internal control [25 (link)]. The relative expression of each miRNA after normalization against hsa-miR-93-5p was calculated according to the following formula: 2 -[Ct (miRNA) - Ct (hsa-miR-93-5p)].
6
Quantifying miRNA Expression by qRT-PCR
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Quantification of miRNAs was performed through a two-step reaction process: reverse transcription (RT) and PCR. cDNA synthesis was carried out by using a reverse transcriptase kit (TIANGEN; catalog number: KR211, China). miRNAs were quantified using SYBR Green (TIANGEN; catalog number FP411, China), as previously described37 (link). The qRT-PCR reactions were carried out by heating at 95 °Cfor 1 min, followed by 40 cycles of 95 °C for 10 sec, 60 °C for 30 sec, and 70 °C for 10 sec. Duplicate assays were performed for each sample.
7
Quantifying HIV-1 Transcript Levels
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Total cellular RNA was extracted by TRIzol reagent (Invitrogen), and reverse transcribed into cDNA using a reverse transcriptase kit (Tiangen, Beijing, China). Real-time PCR was performed using the Thunderbird SYBR qPCR Mix (11203ES08; Yeasen) on the ABI QuantStudio 6 flex Real-Time PCR system. The primers were used as follows.
Intermediate (Int) primers targeted base pair 836–1015 of the HIV-1 transcript, forward, 5′- GTA ATA CCC ATG TTT TCA GCA TTA TC-3′ and reverse, 5′- TCTGGCCTG GTG CAA TAGG-3′;
Distal (Dis) primers targeted base pair 2341–2433 of HIV-1 transcript, forward, 5′- GAG AAC TCA AGA TTT CTG GGA AG-3′ and reverse, 5′- AAA ATA TGC ATC GCC CAC AT-3′.
GAPDH: forward, 5′-ATC CCA TCA CCA TCT TCC AGG-3′ and reverse, 5′-CCT TCT CCA TGG TGG TGA AGA C-3′;
Gag: forward, 5′-GTG TGG AAA ATC TCT AGC AGT GG -3′ and reverse, 5′-CGC TCT CGC ACC CAT CTC-3′;
Intermediate (Int) primers targeted base pair 836–1015 of the HIV-1 transcript, forward, 5′- GTA ATA CCC ATG TTT TCA GCA TTA TC-3′ and reverse, 5′- TCTGGCCTG GTG CAA TAGG-3′;
Distal (Dis) primers targeted base pair 2341–2433 of HIV-1 transcript, forward, 5′- GAG AAC TCA AGA TTT CTG GGA AG-3′ and reverse, 5′- AAA ATA TGC ATC GCC CAC AT-3′.
8
HMGB1 mRNA Expression Analysis
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To detect the expression of HMGB1 mRNA, total RNA was extracted from the stably transfected cells using a RNAprep Pure Cell/Bacteria Kit (Tiangen, Beijing, China; cat: DP430), and cDNA was synthesized using a reverse transcriptase kit (Tiangen, Beijing, China; cat: KR116-02). The HMGB1 forward and reverse primers were 5′-ATATGGCAAAAGCGGACAAG-3′ and 5′-GCAACATCACCAATGGACAG-3′. The β-actin forward and reverse primers were 5′-TGGCACCCA GCACAATGAA-3′ and 5′-CTAAGTCATAGTCCGCCTAG AAGCA-3′. The melting curve data were analyzed to determine PCR specificity. Relative fold expressions were analyzed using the 2−ΔΔCt method, using β-actin Ct values as the internal reference in each sample.
9
Plasma miRNA Extraction and Quantification
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Fasting blood samples (5 mL) were collected in EDTA and separated by centrifugation at 3000g for 15 min. Total RNA containing miRNAs was isolated from plasma using the miRNeasy serum/plasma kit (TIANGEN: catalog number DP503, China). The homogenate was incubated for 5 min at room temperature, 25 fmol of synthetic cel-miR-39 (TIANGEN; catalog number: CD200-01, China) was spiked in. Subsequently, the RNA was extracted according to the manufacturer’s protocols. Total RNA was eluted in 30 µL of RNase-free water. RNA was reverse transcribed to cDNA with reverse transcriptase kit (TIANGEN; catalog number: KR211, China). The reaction system contained total RNA 2 µg, miRNA RT reaction buffer 10 µL, Enzyme Mix 2 µL, RNase-free water up to 20 µL. The mixture was incubated at 42 °C for 60 min, 95 °C for 3 min, and then held at 4 °C. A no-RT negative control was included in each experiment to ensure that PCR products were not due to contamination by genomic DNA.
10
Quantifying Chondrocyte Gene Expression
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Total RNA was extracted from the cultured chondrocyte monolayers through the RNAiso Plus reagent, according to the manufacturer’s instructions. cDNA was obtained through a reverse transcriptase kit (TianGen Biotechnology, Beijing, Co., Ltd. China) with gDNA remover. Real-time quantitative PCR was performed in duplicate to determine the relative gene expression of MMP-3 and MMP-13, with an endogenous control of glyceraldehyde-3phosphate dehydrogenase (GAPDH). Primer sequences (Sangon Biotech, Shanghai, Co., Ltd., China) are provided in Table 1 .
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