The total RNA extraction kit (Invitrogen, CA, USA) was utilized to extract the total RNA in myocardial tissues. The primers (Supplementary Table S1) were compounded by Invitrogen. U6 and β-actin served as the loading control. Revert Aid First Strand Complementary DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) or Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan) was used to reversely transcribe complementary DNA (cDNA) for mRNA or miRNA. The 2−ΔΔCt was used for quantitative analysis. Each reaction was run in triplicate.
Total rna extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Total RNA Extraction Kit is a tool designed to isolate and purify total RNA from a variety of biological samples. It utilizes a simple and efficient method to extract high-quality RNA for downstream applications such as RT-PCR, Northern blotting, and microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cdna synthesis kit, by Thermo Fisher Scientific (7 mentions)
T4 dna ligase, by Promega (6 mentions)
Pfu polymerase, by Promega (4 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by Qiagen (2 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Restriction endonuclease, by Promega (2 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
One step rt pcr kit, by Thermo Fisher Scientific (2 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
Pfu dna polymerase, by Promega (2 mentions)
Taqman mir assay protocol, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Abi prism 7500 real time system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Revert aid first strand complementary dna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (1 mentions)
Lightcycler 480 sybr green 1 master kit, by Roche (1 mentions)
55 protocols using total rna extraction kit
1
Myocardial Total RNA Extraction and qRT-PCR Analysis
2
Quantitative Analysis of GCTB mRNA Levels
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Total RNA samples from cultured GCTB cells or mouse tumors were prepared using the total RNA extraction kit (#15596018; Invitrogen, Guangzhou, China), as per manufacturer’s instructions. RNA concentrations were measured using NanoDrop 2000 instrument (Thermo Fisher Scientific, United States). The cDNA for quantitative reverse transcription polymerase chain reaction (qRT-PCR) was subsequently synthesized from 1 μg total RNA using Evo M-MLVRT kit (#AG11706; Accurate Biotechnology, Hunan, China), according to the manufacturer’s instructions. Then, the qRT-PCR assay was performed using the LightCycler 480 SYBR Green I Master Kit (#4887352001-1; Roche, Guangzhou, China), as per the manufacturer’s instructions. The final expression levels of mRNAs were calculated by the standard 2–ΔΔCt method based on at least three biological replicates. The primer sequences used for quantitation are listed in Table 1 .
3
Quantifying Collagen and MMP-1 Expression
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Total RNA from skin tissue and BJ cells was extracted using a Total RNA Extraction kit (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA was reverse-transcribed into cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. SYBR Green PCR kit (Qiagen) was used as the fluorophorem according to the manufacturer's protocol. RT-qPCR was performed using 2 µl cDNA as a template. The thermocycling conditions were 95˚C for 10 min, 95˚C for 15 sec and 60˚ for 30 sec for a total of 40 cycles. β-actin was used as the reference gene and relative expression levels were calculated according to the 2-ΔΔCq method (20 (link)). The sequences of all primers (Shanghai Shenggong Biology Engineering Technology & Services Co., Ltd.) were as follows: Collagen I forward, 5'-CCAGTCACCTGCGTACAGAACG-3' and reverse, 5'-GCCAGTGTCTCCTTTGGGTCC-3'; collagen III forward, 5'-AGGCAACAGTGGTTCTCCTG-3' and reverse, 5'-GAC CTCGTGCTCCAGTTAGC-3', matrix metalloproteinase-1 (MMP-1) forward, 5'-CCGAGATCTCATGCACAGCTTTCCT CCACT-3' and reverse, 5'-CGGTTAACCGTCAATTTTTCC TGCAGTTG-3', α-smooth muscle actin (α-SMA) forward, 5'-CCACCGCAAATGCTTCTAAGT-3' and reverse, 5'-GGC AGGAATGATTTGGAAAGG-3'; and β-actin forward, 5'-GATCATTGCTCCTCCTGAGC-3' and reverse, 5'-CACCT TCACCGTTCCAGTTT-3'.
4
Quantitative Gene Expression Analysis
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Colon cancer cells in each treatment group were collected. Total RNA extraction kit (Invitrogen, Thermo Fisher Scientific, Inc.) was adopted to extract RNA complying with the protocol. β‐actin was taken as an internal reference. The reverse transcription kit (Takara, RR037A) was adopted to reversely transcribe the RNA, while qRT‐PCR kit (Takara, RR820A) was applied for quantitative analysis. Each sample has to be repeated at least three times. In the end, the relative expression was calculated using 2 − ΔΔ C t 1 lists the involved primer sequences.
5
Extraction and Quantification of Total RNA from Exosomes
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Total RNA was isolated from cells using a total RNA extraction kit (K156002, Invitrogen, CA, USA) according to the manufacturer’s guidelines. Total RNA was isolated from BMSCs-exosomes and chondrogenic BMSCs-exosomes using a total exosome RNA isolation kit (4478545, Invitrogen, CA, USA) according to the manufacturer’s guidelines. The RNA concentrations were detected by NanoDrop (Thermo Scientific, MA, USA). About 500 ng RNA was reverse transcribed into cDNA with PrimeScript RT Enzyme Mix (DRR037A, TaKaRaBio, Beijing, China) according to the manufacturer’s guidelines.
6
RPMI-1640 Cell Culture and Transfection
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RPMI-1640 cell basal culture medium was purchased from Invitrogen (Carlsbad, CA, USA), and fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). The total RNA extraction kit and Lipofectamine 2000 transfection reagent were also purshased from Invitrogen (Carlsbad, CA, USA). Si-MRPS30-DT and the negative control (si-NC) were purchased from GenePharma (Shanghai, China). The anti-Jab1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH was purchased from Proteintech (Wuhan, China).
7
Nucleic Acid Manipulation and Bacterial Conjugation
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The nucleic acid manipulations followed the procedures described by Sambrook et al. (1989 ). Conjugation between the Xcc and E. coli strains was performed as described by Turner et al. (1985 ). The restriction endonucleases, T4 DNA ligase and Pfu polymerase were provided by Promega (Shanghai, China). The total RNAs were extracted from the cultures of the Xcc strains with a total‐RNA extraction kit (Invitrogen, Waltham, MA, USA) and cDNA generated using a cDNA synthesis kit (Invitrogen). These kits were used with reference to the manufacturer's instructions. For semi‐quantitative RT‐PCR, the obtained cDNA was diluted and used as a template with selected primers for target genes (Table S3 ).
8
Rat Gingival RNA Expression Analysis
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We collected the gingival tissues of rats and extracted the total RNA by a total RNA extraction kit (Invitrogen, Carlsbad, CA, USA) and determined the concentration and purity of it. Then, we synthesized cDNA by reverse transcription according to the instructions of the reverse transcription PCR kit (TaKaRa, Tokyo, Japan). The following parameters were used to perform qRT-PCR: 95°C for 1 min, 95°C for 40 s, 58°C for 40 s, 72°C for 45 s, performed 35 cycles, and 72°C for 10 min. β-Actin was used as internal control. The relative expression of the target gene was calculated by the 2−ΔΔCt method. The sequence of primers used is shown in Table 1 .
9
miR-512-5p Expression Quantification
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Total RNA was prepared using Total RNA extraction kit (Invitrogen, USA). The expression level of miR-512-5p was determined using a stem-loop-specific primer method as previously described [54 (link)]. The cDNAs were amplified by RT-qPCR using SYBR Premix Ex Taq (Takara, USA). Primers were purchased from Ribobio (Guangzhou, China), and the fold changes in expression were calculated by relative quantification (2−ΔΔCt); U6 RNA was used as an endogenous control. The primer sequences were provided by Ribobio (Guangzhou, China) (Table 2 ).
10
SARS-CoV-2 RNA Extraction and rRT-PCR
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Total RNA was extracted using total RNA extraction kit (Invitrogen) according to the instructions recommended by the manufacturer. And the nucleic acid was eluted in 50 μL of nuclease-free water and stored at −80 °C until use. rRT-PCR was performed using commercial kits (Biogerm, Shanghai, China) according to the manufacturer's instructions. Orf1ab (FAM reporter) and N (HEX/VIC reporter) genes of SARS-CoV-2 were detected. Samples were positive when PCR gave rise to reliable signals (Ct<38) for either or both genes
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