After transfection with CISD2 shRNA, Beclin1 shRNA, or the corresponding Ctrl shRNA for 48 h, total RNA was extracted with TRIzol (Invitrogen, Thermo Fisher Scientific, USA). Then, a 1/5 volume of chloroform was added for extraction and centrifugation to obtain the upper clear liquid phase, and the same volume of isopropanol was then added and stored at −20°C overnight. cDNA was obtained by reverse transcription with a PrimeScript™ RT Reagent Kit (Promega, USA) in the Promega GoScript reverse transcription system (A5000). Real-time PCR analysis was performed using Promega GoTaq® qPCR Master Mix in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA). With 18S rRNA as the internal reference, qPCR was carried out in a 20 μl reaction system. The 2−ΔΔCt method was used to analyze the data. Three complex wells were set up for all reactions, and the experiment was repeated three times.
Primescript rt reagent kit
Manufactured by Promega
Sourced in United States, China
The PrimeScript RT reagent Kit is a set of reagents designed for reverse transcription of RNA into cDNA. It includes a reverse transcriptase enzyme, buffer, and other necessary components for the reverse transcription reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (57 mentions)
Trizol, by Thermo Fisher Scientific (17 mentions)
Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (14 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (11 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (7 mentions)
Sybr premix ex taq, by Takara Bio (5 mentions)
Sybr green master mix, by Thermo Fisher Scientific (5 mentions)
Gotaq qpcr master mix kit, by Promega (3 mentions)
Taqman power sybr green pcr mix, by Thermo Fisher Scientific (3 mentions)
Taqman power sybr green pcr mix kit, by Thermo Fisher Scientific (3 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Sybr green supermix, by Roche (3 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (3 mentions)
Trizol regent, by Thermo Fisher Scientific (3 mentions)
Gotaq qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Mirna first strand cdna synthesis kit, by Sangon (2 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (2 mentions)
Rna nano chip kit, by Agilent Technologies (2 mentions)
Lightcycler 96 lc96 real time pcr platform, by Roche (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
99 protocols using primescript rt reagent kit
1
Quantifying CISD2 and Beclin1 Knockdown
2
Quantitative Analysis of XIST and miR-141
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Total RNAs were isolated from tissues or cultured cells using Trizol reagent (Invitrogen) and subsequently reverse-transcribed into cDNAs using the PrimeScript RT reagent Kit (Promega, Madison, WI, USA) according to the protocol of the manufacturer. Relative gene expression levels of miR-141 and XIST were measured using TaqMan miRNA assay (Applied Biosystems, Foster City, CA, USA) and SYBR Green PCR Kit (Takara Biochemicals, Kyoto, Japan) under the ABI 7900 Fast Real-Time PCR system (Applied Biosystems), respectively. U6 snRNA and GAPDH were used as the internal control for miR-141 and XIST, respectively. The relative quantification of XIST and miR-141 expression levels was achieved by the 2−ΔΔCt method. The primers for qRT-PCR were as follows: XIST – forward, 5′-CTC TCC ATT GGG TTC AC-3′, reverse, 5′-GCG GCA GGT CTT AAG AGA TGA G-3′; GAPDH – forward, 5′-CAC CCACTCCTCCACCTTTG-3′, reverse, 5′-CCACCACCC TGTTGCTGTAG-3′; miR-141 – forward, 5′-AGACCTCACCTGGCCTGTGGCC-3′, reverse 5′-GAACCCACCCGGGAGCCATCTT-3′; U6 – forward, 5′-CTC GCT TCG GCAGCA CA-3′, reverse, 5′-AAC GCT TCA CGA ATT TGC GT-3′.
3
Microglial and Neuronal Gene Expression
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Total RNA was extracted from microglial cells (3 days post transfection), neurons (3 days post exosomal treatment), and microglia-Exos by using TRIzol reagent (Invitrogen, United States) and reverse transcribed to complementary DNA (cDNA) using a PrimeScript™ RT Reagent kit (Promega, United States) based on the manufacturer’s instructions. Reverse transcription of microRNAs was performed using an miRNA First Strand cDNA Synthesis kit (Tailing Reaction, Sangon Biotech, China). The expression of targeted genes or microRNAs was measured using a GoTaq qPCR Master Mix kit (Promega, United States) and a quantitative PCR system (ABI, United States). GAPDH served as the internal reference of P53, and U6 was the internal reference of microRNAs. The relative quantitative expression was analyzed using the 2 (−ΔΔCt) method. The primers used are listed in Supplementary Table S1 .
4
Gene Expression Analysis of Murine Knee Cartilage
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In total 24 knees were harvested from 12 mice for gene transcript analysis. Pooling was performed to obtain a suitable amount of cartilage and each experimental unit was a pool of two compartments. The cartilage collected from each individual knee (including a femur and tibia) was treated as one compartment. Total RNA from cartilage in knee joints of mice was isolated with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First strand cDNA was synthesized from total RNA using the PrimeScript RT Reagent kit (Promega Corporation) according to the manufacturer's protocols. mRNA expression of MMP-9 and MMP-13 was measured on a 7500 Real-Rime PCR system with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.). ACTB was used as a reference gene (18 (link)). Gene-specific primer sequences used in the present study are listed in Table I . The expression levels of genes were calculated using the 2-ΔΔCq method (19 (link)).
5
Quantitative Analysis of miRNA Expression
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Reagents: TaqMan MicroRNA assay kit (provided by Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and PrimeScript™ RT reagent kit, TRIzol, fetal bovine serum and agar medium (Promega Corporation, Madison, WI, USA) and PCR kit (Jianlun Science and Technology Co., Guangdong, China) were used in the current study. Instruments used were: ultra-low-temperature refrigerator (Sanyo, Osaka, Japan), 7500 HT real-time polymerase chain reaction (PCR) detector, and pure water and water purifier (Angel Water, Inc., Barrington, IL, USA).
6
RNA Quality Assessment and Quantitative PCR
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RNA quality was assessed with the RNA Nano Chip kit (Agilent) on an Agilent Bioanalyser and treatment with DNase was performed using the RNase-free DNase set (Qiagen). Five microgram of total RNA from each sample was used to synthesize cDNA with the PrimeScript RT reagent kit (Promega) and provided oligo-dT primers, according to the manufacturer’s instructions. Quantitative PCR reactions were performed with specific primers listed in Supplementary Table 1 and the GoTaq qPCR master mix kit (Promega) using the Mx3005P Stratagene system. Differential expressions of transcripts of interest were calculated in relation to the 36B4 housekeeping transcript.
7
Real-time PCR Gene Expression Analysis
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Total RNA was extracted with TRIzol reagent in accordance with the manufacturer's instructions (Invitrogen, CA, USA). cDNA was synthesised with the PrimeScript RT reagent Kit (Promega, Madison, WI, USA). Real-time PCR was carried out in a total volume of 10 μl, including 8 μl of TaqMan Power SYBR Green PCR Mix (Invitrogen), 0.5 μl of each primer at 25 μM, and 1 μl of cDNA. The quantitative RT-PCR was carried out on the Roche LightCycler® 96 (LC96) real-time PCR platform using the 2-∆∆CT method. Gene expression results were normalized by internal control GAPDH. Each sample was tested in triplicate.
8
Quantitative RNA Expression Analysis
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Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA) and reverse transcription was performed using the PrimeScript RT reagent Kit (Promega, Madison, WI, USA). cDNA was amplified using SYBR Premix EX Taq™ (Takala, Dalian, China). Primers were as follows: LINC00959 forward, 5’-TGCTCCCATCCCTGCCATGT-3’ and reverse, 5’-AAGACAGGAATCTCGGGTGGGC-3’; GAPDH forward, 5’-AGCCACATCGCTCAGACAC-3’ and reverse, 5’-GCCCAATACGACCAAATCC-3’.
9
Quantifying BCL11A Expression via RT-qPCR
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The methods for isolating RNA was described in previous study.18 (link) We used a PrimeScriptRT Reagent Kit for reverse transcription following the manufacturer’s instructions (Promega, Madison, WI). We performed the Real-time PCR on a Bio-Rad CFX100 using a SYBR Green SuperMix kit (Invitrogen, Carlsbad, CA). We used β-actin as a control for each group. The BCL11A and β-actin primer sequences: BCL11A-F 5-CAGCACTTAAGCAAACGGGAAT-3 and BCL11A-R 5-TTGTTTCCGTTTGTGCTCGATA-3; β-actin-F 5-GGACTTCGAGCAAGAGATGG-3 β-actin-R 5-ATCTGCTGGAAGGTGGACAG-3. The primers were purchased from Invitrogen (Shanghai, People’s Republic of China).
10
Quantitative RNA Expression Analysis
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Total RNAs were isolated using Trizol (Solarbio, Beijing, China) and underwent reverse transcription to cDNA using the Promega PrimeScript RT reagent kit (Promega, Madison, WI, USA). Reverse transcription of mRNA and miRNA was performed as previously described [38 (link)]. qPCR analysis was performed on the synthesized cDNA using Talent qPCR PreMix (SYBR Green) (Tiangen, Beijing, China) with the appropriate amplification conditions: 95 °C, 3 min; 95 °C, 5 s; 50–60 °C, 10 s; 72 °C, 15 s; for 40 cycles. The qPCR primers (Table 1 ) were designed using Primer Premier 5 (Premier Biosoft, CA, USA) and synthesized by Sangon Biotech (Shanghai, China). The 2−ΔΔCt method was used to quantify the expression levels and the results were normalized relative to β-actin (for mRNA) and U6 (for miRNA). Each sample was repeated three times.
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