Mouse liver tissue and skeletal muscle were collected and homogenized. RNA was then extracted from the tissue with TRIzol™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and diluted to 1 μg/μL. One microlitre of the diluted RNA solution was used to obtain a cDNA template using a reverse transcription kit (Solarbio Life Sciences, Beijing, China). One microlitre of the cDNA template was mixed with 10 μL of SYBR Green PCR Master Mix, 1 μL of upstream and downstream primers [see Table 1 (Thermo Fisher Scientific, Inc.)], and 7 μL of sterile distilled water. The mixture was reacted at 95°C for 60 s; then at 95°C for 40 cycles of 15 s each; then at 55°C for 30 s; then at 95°C for 30 s, and finally at 55°C for 35 s. Relative gene expression was calculated using the 2−ΔΔCt method (Stepone Plus qPCR instrument, Thermo Fisher Scientific, Inc.), with GAPDH as the internal reference (21 (link), 22 (link)).
Reverse transcription kit
Manufactured by Solarbio
Sourced in China
The Reverse Transcription Kit is a laboratory tool designed for the conversion of RNA into complementary DNA (cDNA). This kit provides the necessary components, including reverse transcriptase enzyme, buffer, and primers, to facilitate the reverse transcription process. The core function of this kit is to enable the synthesis of cDNA from RNA samples, which is a fundamental step in various molecular biology and gene expression analyses.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (1 mentions)
Stepone plus qpcr instrument, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Dh5α competent cells, by Thermo Fisher Scientific (1 mentions)
Gel recovery kit, by Solarbio (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Total rna extraction kit, by Solarbio (1 mentions)
Trizol reagent, by Solarbio (1 mentions)
Primers for mir 449a, by Thermo Fisher Scientific (1 mentions)
Abi 7900ht instrument, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
7 protocols using reverse transcription kit
1
Quantitative Gene Expression Analysis in Mouse Tissues
2
Cloning tLyp-1-lamp2b Fusion Protein
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Total RNA was extracted from Hela cells using total RNA extraction kit, which was reversely transcribed into cDNA using reverse transcription kit (Solarbio, Beijing, China). tLyp-1 + lamp2b gene was amplified by PCR using cDNA as template, purified and recovered by using gel recovery kit (Solarbio, Beijing, China). The pEGFP-C1 and tLyp-1-lamp2b genes were digested by Bgl II and BamH I enzymes, then connected by T4 ligase. The connection products were transformed into DH5α competent cells (stored at −80 °C, Thermo Scientific, Waltham, USA) and cultured for 12 h. Single colony was culled for plasmid extraction. Lipofectamine 3000 transfection reagent (Invitrogen, USA) were used to transfect pEGFP-C1 plasmid vector expressing tLyp-1-lamp2b fusion proteins into HEK293T cells.
3
qRT-PCR Analysis of CCAT1 and miR-181a-5p
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qRT-PCR was performed as previously described [33 (link)]. Briefly, reverse transcription was performed with the reverse transcription kit (Solarbio, Shanghai, China). Real-time fluorescence PCR was performed using SYBR Green quantification kit (Solarbio). The levels of CCAT1 and miR-181a-5p were normalized to GAPDH mRNA using the 2-ΔΔCt method. PCR primers were designed as Table 4 .
4
Investigating BMSC-derived miR-449a in Wistar Rats
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A total of 55 healthy Wistar female rats weighed from 200 to 250g and aged 7 to 8 weeks were selected. They were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., the animal production license number is SYXK (Shandong) 2017-0001, and the certificate number is 37009200009757. BMSCs from Wistar rats were purchased from Cyagen Biosciences. Sodium pentobarbital, parenzyme, F12 complete media and chloroacetaldehyde hydrate were purchased from Aladdin. TRIzol reagent and reverse transcription kit were purchased from Solarbio. Primers for miR-449a were purchased from Applied Biosystems. BCA protein concentration detection kit and polyacrylamide gel were purchased from Applygen, Beijing. Polyclonal goat anti-rabbit antibodies, IκBα, p50 and p65 antibodies were purchased from Cell Signaling Technology. TNF-α and IL-1β antibodies were purchased from ThermoFisher, China.
5
RNA Extraction and qPCR Analysis
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Extraction of total RNA from peripheral blood was conducted with TRIzol (cat. no. 15596-028; Beijing Solarbio Science & Technology Co., Ltd.) and RNA was converted to cDNA using a reverse transcription kit (cat. no. R202; EnzyArtisan Biotech Co., Ltd.) according to the manufacturer's instructions. Subsequently, qPCR was conducted utilizing 2X S6 Universal SYBR qPCR Mix (cat. no. Q204; Xinbei) on an ABI 7900HT instrument (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for 3 sec, and annealing and extension at 60˚C for 10 sec. β-actin acted as an internal control. The primer sequences are listed in Table I . The mRNA fold variation was calculated with the 2-ΔΔCq method (25 (link)).
6
Quantifying Ptgs1/ptgs2 Gene Expression
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Total RNA was extracted using the Animal Tissue/Cell Total RNA Extraction Kit (DAKEWE, Beijing, China) and cDNA was acquired using a reverse transcription kit (Solarbio, Beijing, China). Ptgs1/ptgs2 transcript levels were measured using an RT-PCR SYBR Green I kit (Solarbio). RT-qPCR was performed on a ROCHE LightCycler® 96 (Roche, Basel, Switzerland) in a 96-well plate, and each sample was prepared in triplicates. The following primer pairs were used: GAPDH, forward primer 5′-TGGCCTTCCGTGTTCCTAC-3′ and reverse primer 5′-GAGTTGCTGTTGAAGTCGCA-3′; mouse COX1, forward primer 5′-TTACTATCCGTGCCAGAACCA-3′ and reverse primer 5′-CCCGTGCGAGTACAATCACA-3′; mouse COX2, forward primer 5′-AGCAAATCCTTGCTGTTCCAA-3′ and reverse primer 5′-GCAGTAATTTGATTCTTGTC-3′.
7
Quantitative RT-PCR Analysis of Mouse Inflammatory Genes
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Next, we extracted total RNA from various cells and spinal cord tissues. SiRNAs from Exo or cells were extracted by a miRNA extraction kit (Solarbio, China). Target mRNA or siRNA was used as template, and the total RNA reverse-transcribed into cDNA using a reverse transcription kit (Solarbio, China). The volume of the whole reaction system was 15 μL. Gapdh was adopted as an internal reference gene for relative quantification by the 2−∆∆Ct method. All primer sequences of mouse gene used in qRT-PCR are shown in the Table 3 .
The primer sequences of mouse gene used in qRT-PCR
| Mouse gene | Primer sequence |
|---|---|
| IL-6 | F:5′-GGTGCCCTGCCAGTATTCTC-3' R:5′-GGCTCCCAACACAGGATGA-3' |
| TNF-α | F:5′-AAGCCTGTAGCCCACGTCGTA-3' R:5′-GGCACCACTAGTTGGTTGTCTTTG-3' |
| Arg-1 | F:5′-CTCCAAGCCAAAGTCCTTAGAG-3' R:5′-GGAGCTGTCATTAGGGACATCA-3' |
| CD206 | F:5′-AAACACAGACTGACCCTTCCC-3' R:5′-GTTAGTGTACCGCACCCTCC-3' |
| GAPDH | F:5′-GTGCTGAGTATGTCGTGGAGTCT-3′ R: 5′-GTGGAAGAATGGGAGTTGCTGT-3′ |
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