In this process, we firstly isolated total RNAs by RNAsimple Total RNA kit (DP419, TIANGEN, Beijing, China) from the whole ovaries of rats or HOMECs. Following measuring the concentrations of extracted RNAs using a NANO 2000 ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), the RNA samples were conducted to be reverse-transcribed into cDNAs by the use of M-MLV reverse transcriptase (NG212, TIANGEN). All designed primers in Table 1
M mlv reverse transcriptase
Manufactured by Tiangen Biotech
Sourced in China
M-MLV reverse transcriptase is an enzyme used in the process of reverse transcription. Its core function is to catalyze the conversion of single-stranded RNA molecules into complementary DNA (cDNA) sequences.
Automatically generated - may contain errors
Lab products found in correlation
Rnasimple total rna kit, by Tiangen Biotech (9 mentions)
Sybr green, by Solarbio (7 mentions)
Dntps, by Tiangen Biotech (3 mentions)
Exicycler 96, by Bioneer (3 mentions)
Taq pcr mastermix, by Tiangen Biotech (3 mentions)
Rnase inhibitor, by Tiangen Biotech (2 mentions)
Methanol, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Rnase free dnase, by Tiangen Biotech (2 mentions)
Trizol reagent, by Transgene (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Nano 2000 ultraviolet spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green reagent, by Solarbio (1 mentions)
Exicycler 96 pcr system, by Bioneer (1 mentions)
Sybr green kit, by Thermo Fisher Scientific (1 mentions)
Pcr master mix kit, by Tiangen Biotech (1 mentions)
Mastercycler real time pcr machine, by Eppendorf (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Random primers, by GenScript (1 mentions)
26 protocols using m mlv reverse transcriptase
1
Gene Expression Analysis in Rat Ovaries
2
Quantification of miRNA and mRNA in TSCC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs in TSCC cell lines were extracted with RNAsimple Total RNA Kit (DP419, TIANGEN, Beijing, China) and reverse-transcribed into cDNA templates using M-MLV reverse transcriptase (NG212, TIANGEN). The designed specific primer sequences were synthesized by Sangon Biotech (Shanghai, China) and shown as follows (5′–3′): miR-135a-5p, RT GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTCACAT, forward GCCGTATGGCTTTTTATTCCTA and reverse GGTGCAGGGTCCGAGGTATT; U6, RT GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAAAATATGG, forward GCTTCGGCAGCACATATACT and reverse GGTGCAGGGTCCGAGGTATT; DANCR forward ACCCTCCTGCTTCCCTC and reverse CCCGAAACCCGCTACAT; KLF8 forward TCATTGGAGGAGATGGTAA and reverse GCTGCTGGTTCTTGCTGT; GAPDH forward GACCTGACCTGCCGTCTAG and reverse AGGAGTGGGTGTCGCTGT. Subsequently, the mixture of cDNA templates, specific primers, SYBR Green reagent (SY1020, Solarbio, Beijing, China) and Taq PCR MasterMix (KT201, TIANGEN) were used to amplify target genes by qRT-PCR analysis on Exicycler 96 PCR system (Bioneer, Daejeon, Korea). GAPDH was normalized for DANCR and KLF8 expression, and U6 was normalized for miR-135a-5p expression. Relative expression was calculated using the 2− ΔΔCT method.
3
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from frozen surgical specimens and UC cell lines using Trizol reagent (Invitrogen, Carlsbad, USA). it was reverse transcripted into cDNA by random primers and M-MLV reverse transcriptase (Tiangen, Beijing, China). RNA quality was evaluated by electrophoresis on a 1% agarose gel. PCR Master Mix kit (Tiangen, Beijing, China) was used for cDNA amplification, and quantitative real-time PCR was performed by a Mastercycler real-time PCR machine (Eppendorf, Hamburg, Germany) using SYBR Green kit (Applied Biosystems, CA, USA) according to the manufacturer’s instruction. The relative mRNA expression levels were analyzed using the 2–ΔΔCt method. All sequences (Table 2 ) were achieved by Primer Premier 5.0 software and GAPDH acted as an internal control.
Sequences of primers and siRNA oligonucleotides
| Primers | Forward primer (5'-3') | Reverse primer (5'-3') | Length of PCR products (bp) |
|---|---|---|---|
| PPARγ | TAGTCGAGGCACCTAGAGA | CTTGTGAATGGAATGTCTTCG | 122 |
| RXRα | TGACGTGCGACGTCGACAA | ACCTTGAGGACGCCATTGAG | 110 |
| GAPDH | GAAGGTGAAGGTCGGAGTC | GAAGATGGTGATGGGATTTC | 226 |
| siRNA | Sense oligonucleotide (5'-3') | Antisense oligonucleotide (5'-3') | Target gene sequence (5'-3') |
| LEF1 | AAGAGAAAGAGAAGUUUGCC | GCAAACUUCUCUUUCUCUUCC | TGGCAAACTTCTTTCTCTTCT |
| Negative control | GUACCGCACGUCAUUCGUAUC | UACGAAUGACGUGCGGUACGU |
4
Quantification of ANRIL Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells using the RNAsimple Total RNA kit (Tiangen Biotech Co., Ltd.). Total RNA was reverse transcribed into cDNA using oligo (dT)15, random primers (GenScript), M-MLV reverse transcriptase (Tiangen Biotech Co., Ltd.), dNTPs, 5X buffer and RNase inhibitor (Tiangen Biotech Co., Ltd.) at 25°C for 10 min, 42°C for 50 min and 80°C for 10 min. qPCR was subsequently performed to analyze the expression levels of ANRIL using cDNA samples, primers (GenScript), SYBR Green (Beijing Solarbio Science & Technology Co., Ltd.) and 2X Taq PCR MasterMix (Tiangen Biotech Co., Ltd.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 94°C for 5 min; followed by 40 cycles at 94°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec; and final extension at 72°C for 150 sec. The following primer pairs were used for the qPCR: ANRIL forward, 5′-CTCCAGACAGGGTCTCACTC-3′ and reverse, 5′-CTGTGTGTCTCCACACTAAG-3′; and GAPDH forward, 5′-GACCTGACCTGCCGTCTAG-3′ and reverse, 5′-AGGAGTGGGTGTCGCTGT-3′. The 2−ΔΔCq method was used to quantify the relative expression levels of ANRIL (24 (link)). GAPDH was used as the loading control.
5
Chemical Reagents for RNA Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In our study, chemicals and reagents used were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). In addition, Oligo (dT), dNTPs, and M-MLV reverse transcriptase were collected from Tiangen (Beijing, China).
6
Quantitative RT-PCR Analysis of COX-2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNAs were isolated from brain tissue by using RNAsimple Total RNA Kit (TIANGEN, Beijing,
China) and added with RNase inhibitor (TIANGEN). Reverse transcription was performed by
using M-MLV reverse transcriptase (TIANGEN). The primers (GenScript, Nanjing, China) used
in this assay were: COX-2, forward 5’- GAACACGGACTTGCTCACTT-3’, reverse
5’- ACGATGTGTAAGGTTTCAGG-3’; GAPDH(internal control), forward
5’-CGGCAAGTTCAACGGCACAG-3’, reverse 5’-CGCCAGTAGACTCCACGACAT-3’. qRT-PCR was carried out
by using SYBR Green (Solarbio, China). The relative gene expression data were calculated
using 2-ΔΔCt method [34 (link)].
China) and added with RNase inhibitor (TIANGEN). Reverse transcription was performed by
using M-MLV reverse transcriptase (TIANGEN). The primers (GenScript, Nanjing, China) used
in this assay were: COX-2, forward 5’- GAACACGGACTTGCTCACTT-3’, reverse
5’- ACGATGTGTAAGGTTTCAGG-3’; GAPDH(internal control), forward
5’-CGGCAAGTTCAACGGCACAG-3’, reverse 5’-CGCCAGTAGACTCCACGACAT-3’. qRT-PCR was carried out
by using SYBR Green (Solarbio, China). The relative gene expression data were calculated
using 2-ΔΔCt method [34 (link)].
7
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 extracted from apical developing leaves using the Trizol reagent (TIANGEN, China) and treated with RNase-free DNase I (Sigma-Aldrich). First strand cDNA was synthesized using 2–5 μg of total RNA with oligo-d(T) primer and M-MLV reverse transcriptase (TIANGEN, China). Real time RT-PCR was performed using SYBR Green-based real-time PCR, a 10 μL reaction mixture containing 5 μl Power SYBR Green PCR Master Mix (2×) (Life, USA), 0.1 μL of each 20 μM primer and 0.3 μL, 60 ng/μL templet were chosen to amplify target sequences. PCR amplification procedures: the first step: 95 °C to 5 minutes. The second step: 95 °C for 5 seconds, 60 °C for 30 seconds, 40 cycles. Third step: from the beginning of 65 degrees, every 5 seconds to increase the temperature of 0.5 degrees, until the end of the reaction at 95°C. eIF4a was used as internal control for N. benthamiana for normalization. The values were calculated using the comparative normalized Ct method [66 (link)] and all the experiments were repeated at least three times. Data were analyzed and plotted with Origin 8.1.
8
Quantitative PCR of RNA Expression
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 reagent (Carlsbad, CA, Invitrogen, USA). One micro-gram of RNA was reverse transcribed to cDNA using the M-MLV Reverse Transcriptase (Tiangen, Beijing, China) according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Mixture (CWBIO, Beijing, China), and the fluorescence signal was detected by iQ5 Multicolor real-time (RT)-PCR system (Bio-Rad, Hercules, CA, USA). PCR primer for human was as follows: glyceraldehyde-3-phosphate dehydroge-nase (GAPDH), 5′-GAAGGTGAAGGTCGGAGTC-3′ (sense), 5′-GAAGATGGTGATGGGATTTC-3′ (antisense); miR-3960 stem-loop, 5′-GTATCCAG TGCAGGGTCCGAGGTATTCG CACTGGATACGACCCCCCG-3′; miR-3960 forward, 5′-ATATATAGGCGGCGGCGGA-3′; universal reverse for miRNA, 5′-GTGCAGGGTCCGAGGT-3′; U6, 5′-CTCGCTTCGGCAGCACA-3′ (sense), 5′-AACGCTTCACGAATTTGCGT-3′ (antisense); HOTAIRM1, 5′-CCCACCGTTCAATGAAAGATG-3′ (sense), 5′-TCAAACACCCACATTTCAACC-3′ (antisense); HOXA1, 5′-CAAAAGAAACCCTCCCAAAAC-3′ (sense), 5′-CGTCAGGTACTTGTTGAAG-3′ (antisense). GAPDH was used as an endogenous control. Fold changes were calculated using the 2−ΔΔCt method. PCRs were conducted in triplicate for each sample. All the reactions were performed in triplicate.
9
Apoptosis Pathway Analysis via DPPH and Trypan
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DPPH and Trypan blue dye, DAPI, Methanol (Sigma, USA); BHT and DMSO (Merck, Germany); M-MLV reverse transcriptase, 1st strand buffer, dNTPs, and primer of oligo (dT), p53, Bax, caspase-3, Bcl-2 and β-actin (Tiangen Biotech, Beijing, China).
10
Quantitative RT-PCR Analysis of Inflammatory Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were isolated by using RNAsimple Total RNA Kit (TIANGEN BIOTECH, Beijing, China). cDNAs were synthesized by M-MLV reverse transcriptase (TIANGEN BIOTECH, Beijing, China). The sequences f primers used in this experiment were shown in Table 1 . Quantitative RT-PCR was performed using SYBR Green (Solarbio, Beijing, China) on ExicyclerTM 96 (BIONEER, Taejon, Korea). The data were analyzed using the 2−ΔΔCT method.
The Detail Sequences of Primers Used for qRT-PCR
| Gene Name | Sequence (5ʹ-3ʹ) |
|---|---|
| MUS TNF-α F | CAGGCGGTGCCTATGTCTCA |
| MUS TNF-α R | GCTCCTCCACTTGGTGGTTT |
| MUS IL-1β F | CTCAACTGTGAAATGCCACC |
| MUS IL-1β R | GAGTGATACTGCCTGCCTGA |
| MUS IL-6 F | ATGGCAATTCTGATTGTATG |
| MUS IL-6 R | GACTCTGGCTTTGTCTTTCT |
| MUS GAPDH F (internal control) | TGTTCCTACCCCCAATGTGTCCGTC |
| MUS GAPDH R (internal control) | CTGGTCCTCAGTGTAGCCCAAGATG |
| HOMO GAPDH F (internal control) | GACCTGACCTGCCGTCTAG |
| HOMO GAPDH R (internal control) | AGGAGTGGGTGTCGCTGT |
| mmu-miR-370-3p F | GCCTGCTGGGGTGGAACCTGGT |
| mmu-miR-370-3p R | GCAGGGTCCGAGGTATTC |
| U6 F (internal control) | CGCAAGGATGACACGCAAAT |
| U6 R (internal control) | GCAGGGTCCGAGGTATTC |
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!