Total RNA was isolated using TRIzol-A+ RNA isolation reagent (Tiangen) following the manufacturer’s protocol. A 0.5 μg aliquot of each sample was treated with DNase to avoid DNA contamination, and then was reversely transcribed using the ReverTra Ace qPCR RT Kit (catalog #FSQ-101, TOYOBO). Samples of acceptable fulfilled the following criteria: OD260/280 ≥ 1.8, OD260/230 ≥ 1.8, 28S/18S ≥ 1.5. The reaction was incubated for 15 min at 37 °C followed by 5 min at 98 °C. Quantitative real time RT-PCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche). The primer sequences used as follows: ILK forward primer, 5'CTTCTGTGGGAACTGGTGACAC3', and reverse primer, 5'GCACAATCATGTCAAACTTGG3'; Actin forward primer: 5'CGTTGACATCCGTAAAGACCTC3' and reverse primer 5'CCACCGATCCACACAGAGTAC3'. Each sample was assayed in duplicate and the levels of mRNA were normalized for each well to the levels of beta-actin mRNA using the 2−△△CT method.
Trizol a rna isolation reagent
Manufactured by Tiangen Biotech
TRIzol-A+ is a reagent designed for the isolation and purification of RNA from various biological samples. It is a mono-phasic solution of phenol and guanidine isothiocyanate that effectively dissolves cells and tissues, while maintaining the integrity of the RNA.
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace qpcr rt kit, by Toyobo (3 mentions)
Sybr green, by Roche (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
Sybr green reagent, by Tiangen Biotech (1 mentions)
Myiq2, by Bio-Rad (1 mentions)
5 protocols using trizol a rna isolation reagent
1
Quantitative RT-PCR for ILK Expression
2
Quantifying HDAC7 mRNA in Brain Regions
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Total RNA was extracted from NAc, cerebral cortex, and dorsal hippocampus using TRIzol-A+ RNA isolation reagent (TIANGEN) according to the manufacturer's protocol. cDNA was then synthesized using the ReverTra Ace qPCR RT Kit (catalog #FSQ-101; TOYOBO). Afterward, real-time qPCR was performed using an SYBR Green PCR kit (Applied Biosystem, USA). The primers were as follows: HDAC7, 5′-GCCTCCATCGACCACTTAACC-3′ (forward) and 5′-CGAGGGTATCTGTCGCAGTC-3′ (reverse); and β-actin, 5′-CGTTGACATCCGTAAAGACCTC-3′ (forward) and 5′-CCACCGATCCACACAGAGTAC-3′ (reverse). β-actin was used as an internal control. The relative expression of HDAC7 mRNA was calculated using the 2−ΔΔCT method as previously described (30 (link)).
3
Quantitative RT-PCR of Metabolic and Inflammatory Genes
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After treatment, cells were used to extract RNA with TRIzol-A+ RNA isolation reagent (TIANGEN). The total RNA was applied to synthesized cDNA by the Revert Aid First Strand cDNA Synthesis Kit (Fermentas). All protocols were performed according to the manufacturer’s instructions. Quantitative RT-PCR was performed in a Cycler (Bio-Rad) using SYBR Green reagent (TIANGEN) to quantification. The β-actin mRNA levels were used to normalization according to 2-ΔΔCT method. The primer sequences used were listed in Table 1 .
Sequence of primers used for quantitative real-time PCR
| Symbol | GenBank | Forward primer (5′-3′) | Reverse primer (5′-3′) |
|---|---|---|---|
| iNOS | NM_001313921 | GAGCGAGTTGTGGATTGTC | CTGCCTATCCGTCTCGTC |
| Arg1 | NM_007482 | ATCTGCCAAAGACATCGT | CATCACCTTGCCAATCCC |
| MCT1 | NM_009196 | GAGGTCCTATCAGCAGTATCTT | CCAGTGGTCGCTTCTTGT |
| MCT2 | NM_011391 | CACTGGCTCCTTTCAATC | CTGGCTTTCTTCAGAGTTG |
| MCT4 | NM_001038653 | ACTTCAACAAGCGTCGCCCTAT | TCAGTCCCTCCGCCTACCTG |
| PFKFB3 | NM_001177752 | AGCCTCTTGACCCTGATA | TTCTTGCCTCTGCTGGAC |
| IL-1β | NM_008361 | ATTGTGGCTGTGGAGAAG | ATCTCGGAGCCTGTAGTG |
| IL-6 | NM_001314054 | TGCCTTCTTGGGACTGAT | TTGCCATTGCACAACTCTTT |
| TNF-α | NM_001278601 | GGCGGTGCCTATGTCTCA | CCTCCACTTGGTGGTTTGT |
| Hif-1α | NM_001313919 | CATCATCTCTCTGGATTTTG | GAAGAGGGAAACATTACATC |
| β-actin | NM_007393 | CGTTGACATCCGTAAAGACCTC | CCACCGATCCACACAGAGTAC |
4
Quantitative Analysis of BDNF Expression
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Transcription of BDNF was measured by quantitative RT-PCR. Cells were washed with PBS, followed by RNA extraction. Total RNA was extracted with TRIzol-A+ RNA isolation reagent (TIANGEN) according to the manufacturer’s instructions. Reverse transcription was performed with 1 μg of total RNA and a RevertAid First Strand cDNA Synthesis Kit (Fermentas). The primer sequences were as follows: BDNF forward primer: 5′-GAC ATC ATT GGC TGA CAC TTT-3′ and reverse primer: 5′-TAC TGA GCA TCA CCC TGG AC-3′; c-Myc forward primer: 5′-CCA CCT CCA GCT TGT ACC TG -3′ and reverse primer: 5′-GAG CAG AGA ATC CGA GGA CG-3′; and HIF1a forward primer: 5′-TTT TGG CAG CAA CGA CAC AG-3′ and reverse primer: 5′- TGA TTG AGT GCA GGG TCA GC-3′. Primers specific for β-actin were used as a control (forward: 5′-TGA CGT GGA CAT CCG CAA AG-3′ and reverse: 5′-CTG GAA GGT GGA CAG CGA GGT-3′). Quantitative RT-PCR was performed in a cycler (MyiQ2, Bio-Rad) using SYBR green (Roche). The threshold cycle for each sample was chosen from the linear range and converted to a starting quantity by interpolation from a standard curve run on the same plate for each set of primers. BDNF mRNA levels were normalized for each well to β-actin mRNA levels using the 2-ΔΔCT method. Each experiment was repeated three times.
5
Total RNA Isolation and qRT-PCR
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Total RNA was isolated using TRIzol-A + RNA isolation reagent (Tiangen, Beijing, China), following the manufacturer's protocol. A 0.5 μg aliquot of each sample was treated with DNase to avoid DNA contamination and then was reverse-transcribed using the ReverTra Ace qPCR RT Kit (catalog #FSQ-101; Toyobo, Osaka, Japan) according to the manufacturer's instructions. Quantitative real-time RT-PCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche, Basel, Switzerland). The primer sequences used are listed in Table 1. Each sample was assayed in duplicate and the levels of miRNA were normalized for each well to the βactin levels using 2 -ΔΔCT .
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