Total RNA was isolated from tumors frozen in liquid nitrogen using ExtractRNA reagent (Evrogen, Moscow, Russia). Residual genomic DNA in the samples was removed by treatment with DNAse I (NEB) for 30 min at 37 °C. RNA was isolated and cleaned with CleanRNA Standard kit (Evrogen, Moscow, Russia). The efficiency of DNAse I treatment was controlled by Real-Time PCR. Transcription of IN genes was assessed by RT-PCR using OneTube SYBR-RT PCR kit (Evrogen, Moscow, Russia) using primers specific to IN and to murine GAPDH (Supplementary Table S3 ). The RT-PCR reaction was performed on BioRad PCR-machine at 45 °C for 15 min, 95 °C 1 min followed by 40× [95 °C 15 s, 62 °C 20 s] cycles.
Cleanrna standard kit
Manufactured by Evrogen
Sourced in China
The CleanRNA Standard kit is a laboratory equipment designed to extract and purify RNA from various biological samples. It utilizes a column-based method to efficiently remove impurities and contaminants, allowing for the recovery of high-quality, intact RNA suitable for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Extractrna reagent, by Evrogen (3 mentions)
Dnase, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Pcr machine, by Bio-Rad (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Sybr green 1, by Evrogen (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Mmlv rt kit, by Evrogen (1 mentions)
Hematoxylin and eosin, by BioVitrum (1 mentions)
Mmlv kit, by Evrogen (1 mentions)
Extractrna, by Evrogen (1 mentions)
Hs taq dna polymerase, by Evrogen (1 mentions)
Sybr green 1 dye, by Evrogen (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Potassium hexacyanoferrate 2 trihydrate, by Merck Group (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
8 protocols using cleanrna standard kit
1
Quantifying Integrase Gene Expression
2
Extraction and Analysis of Rat Brain mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To extract total mRNA from the whole rat brain extract, 1 ml of ExtractRNA reagent (Evrogen, Russia) was added to the frozen rat brain, and then the brain was rapidly homogenized. Total mRNA from cultured cells was also extracted using ExtractRNA reagent according to manufacturer’s instructions. Then mRNA from the rat brain or cultured cells was treated by DNAse I (Sigma-Aldrich) and purified using CleanRNA Standard kit (Evrogen). Total cDNA was synthesized using MMLV RT kit (Evrogen) in accordance to the manufacturer's protocol. After that real-time PCR was performed with the primers described in the S1 Table , and ready-to-use qPCR mix with SYBR Green I and reference ROX fluorescent dyes (Evrogen). Briefly, 12.5 μL of the diluted cDNA sample produced from 1 μg of total RNA was added to 7.5 μL of the PCR master mix and 5 μL of 5× qPCR mix-HS SYBRHigh ROX (Evrogen). Negative controls contained all the components of the PCR mixture except cDNA and gave no signal. All PCR reactions were performed using DT96 Prime real-time detection thermal cycler (DNA Technology, Russia). Data was analyzed by theΔCt method [32 (link)]. Expression level of the nAChR subunits was normalized to the expression level of the β-actin house-keeping gene.
3
Magnetic Nanoparticles for Biomedical Applications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Magnetic nanoparticles coated with glucuronic acid from Chemicell GmbH (Germany) specified as “50-nm fluidMAG-ARA”; hematoxylin and eosin from Biovitrum (Russia); potassium hexacyanoferrate (II) trihydrate, hydrochloric acid, and formaldehyde from Sigma-Aldrich (USA); K3-EDTA test tubes from Guangzhou Improve Medical Instruments (China); ExtractRNA, CleanRNA Standard kit, MMLV kit, HS Taq DNA Polymerase, and SYBR Green I dye from Evrogen (Russia) were used in the experiments. All other chemicals were of analytical grade.
4
Quantitative Analysis of Jasmonate Pathway Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-time quantitative reverse transcription PCR was used for the analysis of the expression of selected genes: ALLENE OXIDE SYNTHASE (TaAOS) (GenBank: KJ001800.1), 12-OPDA REDUCTASE 2 (TaOPR2) (TraesCS7B02G311600), and CORONATINE INSENSITIVE 1-LIKE (TaCOI-1) (TraesCS1A02G279100). Gene sequences were obtained from the GenBank and Ensemble Plants databases [78 (link)]. Total RNA from the second leaf of T3 plants at the four-leaf stage was isolated by Extract RNA reagent (Evrogen, Moscow, Russia), and further purified using the Clean RNA Standard kit (Evrogen, Moscow, Russia) with on-column DNase I treatment (Thermo Fisher Scientific, Welhem, MA, USA) to eliminate DNA contamination. RNA was reverse-transcribed using the MMLV Reverse Transcriptase kit (Eurogen, Russia). Real-time quantitative PCR was conducted in 20 µL reactions containing complementary DNA (cDNA) synthesized from 10 ng of total RNA, SYBR Green I qPCR Master Mix, (Syntol, Moscow, Russia), and 200 nM for each primer. Amplification was performed using Roche LightCycler® 96 System according to the manufacturer’s instructions. TaWIN1 and TaUbi genes were used as reference genes for the internal controls, as previously described for transcript normalization [26 (link)]. Primers are listed in Table S2 .
5
Isolation and Purification of Total RNA from Drosophila Larvae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from 15 larvae of Oregon R flies (modENCODE stock #25211) using RNAzol RT (Molecular Research Center, Cincinnati, OH, USA) in accordance with the manufacturer’s recommendations. The isolated RNA was incubated with 3 U of DNase I (ThermoFisher Scientific, Waltham, MA, USA) for 30 min at 37 °C. The CleanRNA Standard kit (Evrogen, Moscow, Russia) was used for RNA purification. Briefly, 2 μg of total RNA was mixed with 1 μL of the 50 mM oligo(dT)20 primer in a total volume of 13.5 μL, and the mixture was incubated for 5 min at 65 °C. The reverse transcription reaction was carried out in a volume of 20 μL with the following components: 13.5 μL of RNA template with annealed primers, 4 μL of 5× RT buffer (ThermoFisher Scientific), 1 μL of 10 mM dNTP, 1 μL of RNaseOUT (ThermoFisher Scientific), and 100 U RevertAid reverse transcriptase (ThermoFisher Scientific). The mixture was incubated for 60 min at 42 °C, and the enzyme was inactivated for 10 min at 70 °C.
6
Quantitative RT-PCR Analysis of Heart Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was extracted from deep frozen heart apex samples using ExtractRNA™ solution and CleanRNA Standard™ Kit (Evrogen JSC, Russia) according to the manufacturer’s protocols. Reverse transcription (RT) and consequent quantitative real-time PCR (qPCR) were performed using OneTube RT-PCR SYBR™ Kit (Evrogen, JSC, Russia) following the manufacturer’s instructions. RT-qPCR was performed by a Bio-Rad CFX96 thermocycler (Bio-Rad, Hercules, CA). The parameters for RT-qPCR were set as follows: initial heating at 95 °C for 1 min and then followed by 40 cycles with denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and extension at 72 °C for 60 s. Relative expressions of 13 genes of interest and four housekeeping genes were calculated by the 2-ΔΔCt method and normalized by GAPDH, α-tubulin, β-actin and TBP. The primer sequences are listed in Table 2 .
7
RNA Isolation Using TRIZOL Reagent
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using the TRIZOL reagent (Thermo Fisher Scientific, Waltham, MS, USA) according to the protocol provided by the manufacturer. Briefly, the tissue samples were homogenized in TRIZOL. Then, they were subjected to extraction with chloroform (5:1). After centrifugation (12,000× g, 15 min, 12 °C), the upper (aqueous) phase was precipitated with isopropanol (1:1). The pellet was washed in 70% ethanol, spun down (8000× g, 5 min, 8 °C), and dissolved in nuclease-free deionized water (Evrogen, Moscow, Russia). The isolated total RNA was treated with RNase-free DNase I (Qiagen, Hilden, Germany) to remove the traces of genomic DNA. The concentration of RNA was assessed spectrophotometrically with Nanodrop (Thermo Fisher Scientific) equipment according to the instructions provided by the manufacturer. If the A260/A280 ratio in one of the samples did not exceed 2.0, the samples were re-purified with Clean RNA standard kit (Evrogen). The quality of the preparations was verified by electrophoresis in 1.5% agarose gel in non-denaturing conditions.
8
Yeast RNA Isolation and RNA-Seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RNeasy Mini Kit (Quiagen, Germantown, ML, USA) was used for the total RNA isolation from yeast cells. DNA molecules were removed from the sample by DNase (Thermo Fisher Scientific, Waltham, MO, USA) treatment. RNA purification was performed using the CleanRNA Standard Kit (Evrogen, Moscow, Russia). The quality of the isolated RNA molecules was assessed using agarose gel electrophoresis.
RNA-Seq library preparation was performed using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD (Lexogen, Vienna, Austria). The quality of the resulting libraries was tested with the Fragment Analyzer. The pool of libraries was sequenced on an Illumina MiSeq (single read, 300 bp) at Evrogen, Moscow, Russia.
RNA-Seq library preparation was performed using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD (Lexogen, Vienna, Austria). The quality of the resulting libraries was tested with the Fragment Analyzer. The pool of libraries was sequenced on an Illumina MiSeq (single read, 300 bp) at Evrogen, Moscow, Russia.
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!