Total RNA was extracted from the peri-infarct tissues using RNAprep pure Kit (for tissue) (Tiangen, DP431, Beijing, China), and cDNA was synthetized using FastQuant RT Kit (Tiangen, KR106, Beijing, China). RT-PCR was performed on a CFX ConnectTM Real-Time PCR Detection System (Bio-Rad) using SuperReal PreMix Plus (Tiangen, FP205, Beijing, China). The primer sequences were as follows (forward and reverse, respectively): NG2, 5′-AGCCCATGGCC TTCACTATCAC-3′ and 5′-CCGGCCCTGAATCACTGTCTA-3′; CNPase, 5′-GGAGACATAGTGCCCGCAAAG-3′ and 5′-TTGCACTCGTGCAGCGTA-3′; growth associated protein-43 (GAP-43), 5′-CACCATGCTGTGCTGTATGAGAA-3′ and 5′ -GTCCACGGAAGCTAGCCTGA-3′; NogoA, 5′-CTTGGTCA TGTGAACAGCACAATAA-3′ and 5′-CATTGAACAAGGCAC CAACGTAA-3′; NgR, 5′-TCCAGTCATGCCGAAATCTCAC-3′ and 5′-TGGTAGGGTCCACGACATGAAG-3′; RhoA, 5′-CA GCAAGGACCAGTTCCCAGA-3′ and 5′-AGCTGTGTCCCAT AAAGCCAACTC-3′; ROCK2, 5′-CTAACAGTCCGTGGGTGG TTCA-3′ and 5′-CTCAGGCACATCATAATTGCTCATC-3′; β-actin, 5′-GGAGATTACTGCCCTGGCTCCTA-3′ and 5′-GACTCATCGTACTCCTGCTTGCTG-3′. The reactive conditions were as follows: denaturing at 95°C for 15 min, followed by 40 cycles of 95°C for 10 s, 55°C for 31 s, and 72°C for 30 s. Data were presented as relative mRNA level according to the 2–ΔΔCt method. Actin served as an internal standard.
Rnaprep pure kit for tissue
Manufactured by Tiangen Biotech
Sourced in China
The RNAprep Pure kit (For Tissue) is a product designed for the extraction and purification of RNA from a variety of tissue samples. The kit utilizes a specialized protocol and reagents to efficiently isolate high-quality RNA, which can then be used for downstream applications such as reverse transcription and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Fastquant rt kit, by Tiangen Biotech (1 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti wif 1, by Cell Signaling Technology (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Gel doc 1000 system, by Bio-Rad (1 mentions)
Gel pro analyzer software version 3, by Media Cybernetics (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
4 protocols using rnaprep pure kit for tissue
1
Quantifying Gene Expression in Stroke Tissues
2
Tissue RNA Extraction and Analysis
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The RNAprep Pure kit (For Tissue) was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The RevertAid First Strand cDNA Synthesis kit was purchased from Fermentas (Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). A reverse transcription-quantitative polymerase chain reaction (RT-qPCR) kit (DRR01AM) was purchased from Takara Bio, Inc. (Otsu, Japan). Antibodies specific to β-catenin (catalog no., sc-7199) and β-actin (catalog no., sc-130300) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-WIF-1 (catalog no., 5652) and anti-cyclin D1 (catalog no., 1044S) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Peroxidase-conjugated ImmunoPure® goat anti-rabbit immunoglobulin G (IgG) [heavy (H) + light (L)] and biotinylated ImmunoPure® goat anti-mouse IgG (H + L) (catalog no., MII0401) were purchased from Pierce (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All other reagents used were analytical-grade laboratory chemicals from standard commercial suppliers.
3
Quantitative RT-PCR Analysis of Lung Tissues
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Total cellular RNA of lung tissues was isolated using the RNAprep Pure kit (For Tissue) (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. Total RNA (2.0 µg) was reverse transcribed into complementary DNA using the RevertAid First Strand cDNA Synthesis kit with oligo(dT). All primers were designed and synthesized by Takara Bio, Inc. The primer sequences, annealing temperatures and expected product sizes are listed in Table I . All PCR procedures were conducted in the MJ Research PTC-200 DNA Engine Thermal Cycler (Bio-Rad Laboratories, Inc.) using the following cycling parameters: 2 min of initial denaturation at 94°C, followed by 30 sec of denaturation at 94°C, and 35 cycles of 30 sec at 94°C (denaturing), 30 sec at 58°C (annealing) and 30 sec at 72°C (elongation), with a final extension at 72°C for 5 min. The presence of PCR products was confirmed by gel electrophoresis on a 2% agarose gel and staining with ethidium bromide. Bands were visualized in a Gel Doc 1000 system (Bio-Rad Laboratories, Inc.). β-actin was used as an internal control in parallel for each replicate. Gel-Pro Analyzer software version 3.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to quantify the denisty of each band. The experiments were performed three times independently, and the mean value was used for comparison.
4
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted from the tissues using an RNAprep Pure Kit (For Tissue) (Tiangen), according to the manufacturer instructions. Total RNA was quantified by absorbance at 260 nm, and its integrity was checked by agarose gel electrophoresis and ethidium bromide staining of the 28 and 18S bands. Total RNA was reverse-transcribed into cDNA using a PrimeScript ™ RT reagent kit (Takara), according to manufacturer instructions. A real-time PCR was conducted with 12.5 μL SYBR ® Premix Ex Taq™ II (Takara), 0.2 μL 10 μM primers, 9.5 μL ddH 2 O, and a 1-μL total reaction volume that contained 100 ng cDNA. The thermal cycling parameters were as follows: 95°C for 5 s, followed by 40 cycles at 95°C for 30 s and Tm for 30 s. All of the primers were designed using integrated mRNA sequences based on sequences published by the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov; Table 1). The 2-△△Ct method was used to determine the relative fold-changes (Schmittgen and Livak, 2008) , and all of the data were normalized with the housekeeping glyceraldehyde-3-phosphate dehydrogenase gene.
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