Total RNA was purified from cells using TRIzol reagent (Invitrogen) following the manufacturer’s instruction. A Revertaid™ First Stranded cDNA Synthesis Kit (Fermentas) was used to synthesize cDNA from RNA. PCR was performed using an iTaq DNA Polymerase Kit (iNtRON biotechnology) following the manufacturer’s guidelines. The primer sequences used for PCR are as follows: 5’- TGCGGAACCAGAGCAGGGGT-3’ and 5’- TTTGGGCCAGTGTTCCCGCC-3’ for FCGRI (NM_010186.5), 5’- TGGGAGGTCCATCCGGAGCC-3’ and 5’- TCAGGAGGATTGTCTGGAACCTGC-3’ for FCGRIIB (NM_010187.2), 5’-TTCCACCACTGACAATTCTGCTGCT-3’ and 5’-GGCCCGTGTCCACTGCAAACA-3’ for FCGRIII (NM_010188.5), and 5’-GCCAAACGGGTCATCATCTC-3’ and 5’-GACACATTGGGGGTAGGAAC-3’ for GAPDH (NM_008084.2).
Revert aid first stranded cdna synthesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Revert Aid First Strand cDNA Synthesis Kit is a lab equipment product designed to facilitate the reverse transcription process, which converts RNA into complementary DNA (cDNA). The kit provides the necessary components, including an RNA-dependent DNA polymerase, to efficiently generate first-strand cDNA from total RNA or poly(A)+ RNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Itaq dna polymerase kit, by iNtRON Biotechnology (1 mentions)
Rna mini kit, by Favorgen Biotech (1 mentions)
I taq polymerase, by iNtRON Biotechnology (1 mentions)
Megalign, by DNASTAR (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green, by Qiagen (1 mentions)
Ribospin 2 rna extraction kit, by GeneAll (1 mentions)
Lightcycler system, by Roche (1 mentions)
7 protocols using revert aid first stranded cdna synthesis kit
1
Expression Analysis of Fc Gamma Receptors
2
Quantifying Tetracycline Resistance Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-PCR was conducted to determine the expression level of the conserved regions of cmeA in all phenotypically tetracycline-resistant isolates. Briefly, total RNA was extracted using a RNA Mini Kit (Favorgen, Taiwan) according to the manufacturer's instructions, and subjected to cDNA synthesis using a RevertAid™ First Stranded cDNA Synthesis Kit (Fermentas, Lithonia). The 16SrRNA gene was used as an endogenous control. The PCR reaction mixture contained 12.5 µL of SYBR Green Master Mix, 400 ng of template DNA, 0.25 µM of forward and reverse primers each, and 12 µL of nuclease-free water. RT-PCR cycling conditions were as follows: 10 min at 50 °C, 5 min at 60 °C, and 5 min at 95 °C and then 40 cycles of 10 s at 95 °C, 30 s at 58 °C, and 15 s at 72 °C [29 (link), 30 (link)].
3
Eupatilin Modulates Inflammatory Cytokine Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FLS were incubated with 1, 2, 5, or 10 µM eupatilin or DMSO for 24 hr, followed by incubation with TNF-α (10 ng/mL) for 15 min. The cells were harvested, and total RNA was extracted with TRIZOL reagent. cDNA was synthesized using a Revertaid First-stranded cDNA Synthesis Kit (Thermo Fisher Scientific Inc, Pittsburgh, PA, USA). RT-PCR was performed using i-Taq polymerase (iNtRON Biotechnology, Seongnam, Korea) and primers for human IL-6 (sense, 5'-ATG AAC TCC TTC TCC ACA AGC GC-3'; antisense, 5'-GAA GAG CCC TCA GGC TGG ACT G-3'), IL-1β (sense, 5'-ACT GCA CGC TCC GGG ACT CA-3'; antisense, 5'-AAG GGC TGG GGA TTG GCC CT-3'; and GAPDH (sense, 5'-ACC ACA GTC CAT GCC ATC AC-3'; antisense, 5'-TCC ACC ACC CTG TTG CTG TA-3').
4
Comprehensive Molecular Detection of Enteric Pathogens in Piglets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 528 diarrheic samples including feces, fecal swabs and small intestine were collected from 43 pig farms of 10 provinces and cities during 2011–2017, where nearly 100% of the non-weaning neonates died from severe diarrhea (Table S1). And according to our survey, except for several pig farms with no PEDV vaccination, most of the pig farms were immunized by commercial inactivated PEDV vaccines. The total RNA of each sample was extracted by RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed into cDNA by Revert Aid First Stranded cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) as described in the handbooks. Then the cDNA was used for PCR with specific primers designed based on the conserved regions of PEDV, TGEV, porcine deltacoronavirus (PDCoV) and Group A Rotavirus (RoV-A) as described previously(Wang et al., 2016 ). PCR products were analyzed by electrophoresis in 1.0% agarose gels.
5
Genomic Analysis of FJzz1 Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA of FJzz1 variants F20, F50, F100, F150 and F200 was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany), and then reverse transcribed into cDNA with the Revert Aid First Stranded cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), as described previously. PCR was performed with this cDNA as the template and 16 overlapping pairs of primers (Supplementary Table S1). The genomic sequences of the FJzz1 variants were determined with the next-generation sequencing technology. The aa sequences of the S proteins and the full-length genome sequences of these strains were also aligned using the Clustal W method in MegAlign (DNAStar Lasergene), and the N-linked glycosylation sites in the S proteins of these strains were predicted and analyzed with the NetNGlyc 1.0 Server, as described previously (Chen et al. 2019 (link)). A phylogenetic tree was constructed with the neighbor-joining method (NJ) using MEGA 7.0 based on S gene of FJzz1 strain and another 60 reference strains with complete S gene sequences available in GenBank (Supplementary Table S2).
6
Quantification of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To extract total cellular RNA, Ribospin II RNA extraction kit (GeneAll Biotechnology Co., Ltd.) was used following the manufacturers instructions. The total RNA concentration and purity were quantified by applying the NanoDrop spectrophotometer (ND-1000; Thermo Fisher Scientific, Inc.). Complementary DNA was synthesized from 1 µg of total RNA employing RevertAid First Stranded cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturers protocol. RT-qPCR for RNA expression quantification was carried out on Applied Biosystems (StepOnePlus; Thermo Fisher Scientific, Inc.) using QuantiTect SYBR-Green (Qiagen GmbH) with a total reaction volume of 10 µl and the initial denaturation at 95°C for 15 min, followed by 40 cycles of PCR at 95°C for 15 sec, 63°C for 10 sec, 72°C for 20 sec. Comparative analysis by 2(−∆∆Cq) was used to compare the expression of target genes (77 (link)). Relative expression software tool (REST) was applied for comparison (78 (link)), and the statistical analysis of relative expression results in real-time PCR. The related primers (Ki-67, cyclin D1, Rb, p21, BiP and 14-3-3ζ) (Table II ) were obtained from Sigma-Aldrich; Merck KGaA. GAPDH served as the reference gene. All reactions were performed in triplicate.
7
PEDV RNA Expression Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the manufacturer's instructions, total RNA was extracted from PEDV-infected LLC-PK1, ST and MARC-145 cells using the RNeasy Mini kit (QIAGEN, 74104) For reverse transcription (RT)-qPCR analysis, one microgram of total RNA was transcribed to cDNA using the Revert Aid First Stranded cDNA Synthesis Kit (Thermo Fisher Scientific, K1622). The synthesized cDNA was then used as the template for quantitative PCR using SYBR Green PCR mix according to the manufacturer's instructions with a LightCycler system (Roche, Switzerland). The RT-qPCR-specific primers are listed in Table 1
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!