For cDNA synthesis, 1000 ng of intact RNA was reversed to cDNA with a reverse transcription kit (YEASEN, Shanghai, China) with the concentration detected. The synthesized cDNA was diluted to 200 ng/μL as a template for RT-qPCR. To detect carbohydrate metabolism, the expression of genes related to glycogen synthesis (ugp2b, gys2), glycogen degradation (pygl), gluconeogenesis (pck1, pcxb), glycolysis (gck), TCA cycle pathway (idh), and pentose phosphate pathway (g6pd) were evaluated. The expression of genes related to lipid synthesis (fasn, acaca, aclyb) and decomposition (acadl, acaa1, lpl) were determined to illustrate the influence on lipid metabolism, and the expression of genes related to the urea cycle (gs, cps3, otc, ass, asl, and arg1) was also detected. The qPCR was performed in Jena qTOWER3G system using the real-time quantitative PCR detection kit EvaGreen 2 × qPCR Master mix (YEASEN, Shanghai, China): pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 10 s; annealing and extension at 60 °C for 30 s; and PCR reaction step running 40 cycles. After RT-qPCR, melting curves were analyzed to ensure the specificity of the reaction. Using 18s as the internal reference gene, the relative quantitative data analysis was performed by the 2−ΔΔCt method. RT-qPCR data were analyzed using GraphPad Prism 6. The primers used in the present study are shown in Table 1 .
Reverse transcription kit
Manufactured by Yeasen
Sourced in China
The Reverse Transcription Kit is a laboratory tool used for the synthesis of complementary DNA (cDNA) from RNA templates. It contains the necessary reagents and enzymes required for this process, enabling the conversion of RNA into a more stable DNA format for further analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (17 mentions)
Sybr green master mix, by Yeasen (9 mentions)
Trizol reagent, by Takara Bio (4 mentions)
Sybr green kit, by Yeasen (4 mentions)
Trizol reagent, by Yeasen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Agarose gel electrophoresis, by Biosharp (2 mentions)
Dna polymerase, by Vazyme (2 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq, by Yeasen (2 mentions)
Trieasy total rna extraction reagent, by Yeasen (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Qpcr kit, by Yeasen (2 mentions)
Trizol, by Solarbio (2 mentions)
Sybr green pcr kit, by Yeasen (2 mentions)
Hieff qpcr sybr green master mix, by Yeasen (2 mentions)
C1000 touch thermal cycler, by Bio-Rad (2 mentions)
Pcr system, by Bio-Rad (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
62 protocols using reverse transcription kit
1
Comprehensive Gene Expression Analysis of Metabolic Pathways
2
Evaluating EVC Gene Splicing Patterns
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Constructs containing the wild‐type and mutant EVC transcripts were transfected into HeLa and HEK293T cells using Lipofectamine 2000 (Yeasen Biotech). Total RNA was extracted from HeLa and HEK293T cells at 48 h post‐transfection using Trizol Reagent (Takara), and the cDNA was transcribed using a reverse transcription kit (Yeasen Biotech). Minigene‐specific cDNA was amplified using plasmid‐specific primers. Splicing pattern analysis was done via electrophoresis on 2% agarose gels and with Sanger sequencing. The primers for EVC gene amplification are listed in Table S1 .
3
Quantitative Real-Time PCR for Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells by TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed using a Reverse Transcription kit (Yeasen Biotechnology Co. Ltd.), and the resultant cDNA was utilized as the qPCR template. The reaction system of RT-qPCR was 2 μl, composed of 0.4 μl F, 0.4 μl R, 7.2 μl DEPC, 2 μl cDNA and 10 μl of SYBR. Then, RT-qPCR was carried out on a Light Cycler 480 system using SYBR GREEN PCR Master Mix (Yeasen Biotechnology Co. Ltd.) according to the real-time PCR manufacturer’s instructions. U6 and GAPDH served as internal controls for miR-93-5p and mRNA, respectively. The relative levels of genes were calculated using the 2-△△Ct method. Table 1
4
RNA Extraction, cDNA Synthesis, and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA preparation was conducted with TRIzol reagent (10606ES60, Yeasen), and cDNA synthesis was performed according to the instructions on the reverse transcription kit (11141ES60, Yeasen). qPCR was carried out with TB Green® Premix Ex Taq™ (RR820A, TaKaRa) on a CFX Connect™ Real-Time System (CFXConne, Bio-Rad). qPCRs were performed in at least three independent experiments, and relative gene expression was quantified based on the 2−ΔΔCt method with reference genes as a normalization indicated control. For RT–PCR assays, based on ASE analysis, primers for detecting exon skipping were designed in Primer Premier 5 for validation of RNA splicing. RT–PCR was performed with the following program: 95°C for 2 min, 36 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s and elongation at 72°C for 1 min, and a final elongation temperature of 72°C for 5 min and 4°C holding temperature. PCR products were run in 2% agarose gels stained with 0.1‰ ethidium bromide (EB) dye (C14141868, Macklin). Bands were visualized with a GleUV system (Baygene Biotech). Band intensities were quantified in ImageJ software.
5
Quantification of miR-30b-5p and GRIN2A Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from hippocampal neurons using a MolPure Cell RNA Kit (Yeasen, China), according to the manufacturer’s instructions. A reverse transcription kit (Yeasen, China) was used for reverse transcription. The expression levels of miR-30b-5p and GRIN2A were determined using a Real-Time PCR kit (Thermo Fisher, USA) on a real-time fluorescence quantitative PCR system (ABI7500). Relative expression was calculated using the 2−ΔΔCt method by normalizing U6 or GAPDH. Primers used are listed in Table 1 .
6
GPX2 Expression in PCa Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human PCa cell lines (PC-3, DU145, LNCaP, and 22RV1) were purchased from Procell (Wuhan, China). The cells were cultured in RPMI-1640 (bl303a; Biosharp, Anhui, China) at 37 °C and in the presence of 5% CO2. The cells adhered to the wall and were passaged every 3 days. The cells in the logarithmic growth phase were used for the experiment. TRIzol reagent (bs259a, Biosharp, China) was used to extract the total RNA of PC-3, LNCaP, 22Rv1, and DU145 cells. A reverse transcription kit (11119es60; Yeasen, Shanghai, China) was used to reverse transcribe RNA into cDNA, and a SYBR Green Kit (11201es50; Yeasen, China) was used for qRT-PCR amplification, with β -actin as an internal control. The 2- Δ ΔCT method was used for calculation. The primers were as follows: GPX2: 5′-GCCTCCTTAAAGTTGCCATA-3′ and 5′-GCCCAGAGCTTACCCA-3′; β -actin: 5′-GAAGAGA-GAGACCCTCACGCTG-3′, and 5′-ACTGTGAGGAGGGGAGATTCAGT-3′. The experiment was repeated three times.
7
Quantification of mRNA Expression by RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from frozen quadriceps femoris muscle and liver samples or from differentiated primary satellite cells using TRIzol reagent (Invitrogen). A reverse transcription kit (Yeasen, China) was used to synthesize cDNA in a 20 μl reaction containing 1–5 μg of total RNA. A SYBR Green kit (Yeasen, China) was used to perform RT-qPCR in a 20 μl reaction containing 0.5–1 μl cDNA. Target mRNA levels were normalized to that of β-actin as an internal control and changes in expression were calculated using the formula 2-△△Ct. The primer sets used are shown in Supplementary Table 1 .
8
Quantitative Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse Transcription Kit (11141ES10, Yeasen, Shanghai, China) was used to obtain complementary DNA. Then, quantitative PCR (qPCR) was performed using SYBR Green Master Mix (11202ES03, Yeasen, Shanghai, China). The mRNA levels of target genes were normalized to the value of β-ACTIN mRNA in each sample and fold-change in expression was calculated using the 2ΔΔCt method. The oligonucleotide primers (Sangon Biotech, Shanghai, China) for target genes are listed in Table 1 .
9
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from cells using TRIzol according to the manufacturer’s instructions. Reverse transcription was performed using the Reverse Transcription Kit (Yeasen). qRT–PCR was performed using the SYBR Green Master Mix (Yeasen). Primers were listed in Table S2 . Results were analyzed using the 2−(ΔΔCt) method.
10
RNA Quantification via RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell sediments were collected, and RNA sediments were extracted using Trizol lysis (Thermo, USA). RNA samples were reverse transcribed according to the Reverse Transcription Kit (Yeasen, Shanghai, China). The reverse transcribed cDNA samples were diluted 5-10-fold with DEPC and stored at -20 °C. Next, real-time fluorescence quantitative PCR was performed using SYBR Green Master Mix (Yeasen, Shanghai, China) and determined on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, USA). Quantitative expression levels were analyzed using the 2−ΔΔCt method and normalized with β-actin. Primer sequences are shown in Table 1 of supplementary materials.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!