Circulating DNA was extracted by using commercially available kit (Epigentech, USA) following manufacturer’s protocol and circulating total RNA was extracted by Trizol reagent according to the manufacturer’s protocol (AMRESCO, USA) of lung adenocarcinoma cases as well as from healthy control’s circulating samples stored at –80 °C. The quality of circulating DNA and total RNA samples were quantified using NanoDrop spectrophotometer. The ratios of the absorbance at 260 and 280nm (A260/280) were used to assess the purity of nucleic acids and for pure DNA, A260/280=1.8 and for pure RNA A260/280 =2.0 were considered.and from total RNA, cDNA was synthesized by using 100 ng total RNA following manufacturers’ protocol (Verso, Thermo Scientific, USA). Briefly, 100 ng of total RNA, 5X cDNA synthesis buffer, dNTPs (5 mM each), RT enhancer, Verso RT enzyme mix and random hexamers/Oligo DT (400 ng/μL) in the total volume of 20 μL incubated for 60 min at 42°C.
Verso
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Verso is a compact and versatile laboratory centrifuge designed for a wide range of applications. It features a small footprint and delivers efficient sample processing with its high-speed rotation capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Avantor (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dynamo colorflash sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Hipura yeast rna purification kit, by HiMedia (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Custom primers, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by BD (1 mentions)
Il 22, by BioLegend (1 mentions)
Rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green based qpcr, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
14 protocols using verso
1
Circulating DNA and RNA Extraction
2
Quantifying lncRNA H19 Expression in T2DM
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A 100 ng of the total cell-free extracted RNA from T2DM patients and healthy controls were used to synthesize cDNA using kit [Verso, Thermo scientific, USA] following kit provided protocol. Expression of cell-free lncRNA H19 was done by quantitative real-time PCR using SYBR green dye using forward primer sequence 5ʹ-ATCGGTGCCTCAGCGTTCGG-3ʹ and reverse primer sequence 5ʹ-CTGTCCTCGCCGTCACACCG-3ʹ and β-actin were used as internal control, and the forward primer sequence 5ʹ-CGACAACGGCTCCGGCATGTGC-3ʹ, reverse primer sequence 5ʹ-GTCACCGGAGTCCATCACGATGC-3ʹ. The program followed as qRT-PCR for lncRNA H19 and β-actin was performed for 40 cycles; initial denaturation was at 94°C for 40 seconds, annealing temperature was at 60°C for 40 seconds, extension at 72°C for 40 seconds and 20 µl reaction volume was used. Ending additional step at 72°C for 5 minutes to end up reaction and melting curve examined between the ranges 35°C to 90°C to confirm the target amplification. A control without cDNA was included in each experiment, and every reaction was done in duplicate. The relative quantification by 2− [ΔΔCT] method was used to compute the lncRNA H19 level expression.
3
Quantifying Circulating lncRNA Expression
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One hundred nanograms of total circulating RNA extracted from all the study participants was used to synthesize cDNA using a kit (Verso, Thermo Scientific, USA) following the provided protocol. Expression of the circulating lncRNAs NF-kappaB interacting lncRNA (NKILA), nuclear enriched abundant transcript 1 (NEAT1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and myocardial infarction-associated transcript (MIAT) was determined by qRT-PCR using SYBR Green dye with specific primer sequences (online supplemental table 1 ). The following program was used for qRT-PCR to amplify the lncRNAs NKILA, NEAT1, MALAT1, and MIAT and β-actin in a 20 μL reaction volume: 40 cycles of initial denaturation at 94°C for 40 s; annealing for 40 s at 64°C (NKILA), 64°C (NEAT1), 60°C (MALAT1), 60°C (MIAT) or 64°C (GAPDH); and extension at 72°C for 40 s. A final additional step at 72°C for 5 min to end the reaction and melting curve examination between 35°C and 90°C were performed to confirm target amplification were performed. A control sample without cDNA was included in each experiment, and every reaction was performed in duplicate. Relative quantification by the 2−(ΔΔCT) method was used to compute expression levels of the lncRNAs NKILA, NEAT1, MALAT1, and MIAT.
4
Quantitative Analysis of ATG8 and α-SYN
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Quantitative real-time PCR (qRT-PCR) was used to measure the mRNA transcripts of ATG8 and α-SYN. Phoshphoglycerol kinase (PGK1) was selected as a reference gene. Total RNA was isolated from cell lysates using a HiPurA Yeast RNA Purification Kit (HiMedia, India-MB611) following the manufacturer’s protocol. The cDNA was prepared from the isolated RNA using a cDNA synthesis kit (Verso, Thermo Scientific, USA-AB1453B), and used as a template for qRT-PCR. The cDNA samples were amplified with a DyNAmoColorFlash SYBR green qPCR kit (Thermo scientific, USA-FNZ416L) using a Steponeplus Real time PCR system (Applied Biosystems).
5
Quantification of lncRNA LINC01173 Expression
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Of the total extracted RNA from study participants, 100 ng were used to make cDNA using a kit (Verso, Thermo Scientific, USA) according to the kit’s instructions. Expression of lncRNAs LINC01173 was done by quantitative real-time PCR using SYBR green dye using specific primer sequences (forward primer: 5’-CTAGGCTCAGACCTGGCAAC-3’ and reverse primer: 5’-GGCCTTGGGGAAACGAAGAT-3’). The program followed: qRT-PCR for lncRNAs LINC01173, and β-actin was performed for 40 cycles, initial denaturation was at 94°C for 50 seconds, annealing temperature for, LINC01173, and β-actin was at 60°C for 50 seconds, extension at 72°C for 50 seconds, and a 20 µL reaction volume was used. To confirm the target amplification, an additional step at 72°C for 10 minutes was performed to finish the reaction and a melting curve examination was performed between 35°C and 90°C and every reaction was done in duplicate. The lncRNAs LINC01173 expression level was calculated using the relative quantification by 2−(ΔΔCT) method.
6
Quantifying cIAP-1 and cIAP-2 mRNA Expression
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100 ng of total RNA was used to synthesise cDNA, following manufacturer's protocol (Verso, Thermo Scientific, USA). The cIAP-1 and cIAP-2 mRNA expression was done by quantitative RT-PCR using SYBR Green I technology, and the beta-actin gene was used as the housekeeping control to analyse the fold change in mRNA expression. The primer sequences for cIAP-1 and cIAP-2 mRNA and beta-actin mRNA amplification are depicted in Table 1 . The cIAP-1 and cIAP-2 mRNA expression study was executed by using the programme for 40 cycles, with the first denaturation step at 94°C for 35 s, annealing was for 40 s to 600°C with various temperatures, and extension was done at 72°C for 40 s, and the final reaction volume was maintained to 20 μl. The final step for extension was at 72°C for 5 minutes. Melting curve analysis was done between the temperature ranges of 35°C and 90°C for target amplification, and all processes were performed in duplicate to avoid errors. The relative quantification method, 2-(∆∆CT) method, was used to calculate the cIAP-1 and cIAP-2 mRNA expression using beta-actin as the in-house control, and finally, the results were expressed as the mean fold change in breast cancer patients compared to controls.
7
Profiling Cytokine Expression in PCP
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3 ml of peripheral blood samples were withdrawn from suspected cases of PCP as well as from healthy control subjects. Total RNA extraction from the blood sample was executed using the QIAamp DNA Mini Kit (Qiagen, Germany) following instructions. The quality and quantity of total RNA were determined by the A260/280 ratio using the nano-spectrophotometer method.100 ng total RNA was taken to synthesize the cDNA by kit provided-method (Verso, Thermo Scientific, USA) for IL-2, IL-4, IL-10, IL-13 mRNA expression following the manufacturer’s protocol.
8
Isolation and Quantification of IECs
9
Total RNA Isolation from Whole Blood
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RNA Isolation kit (Thermo Fisher Scientific, Massachusetts, USA) was carried out for the total RNA extraction from frozen whole blood using the following manufacturer-provided instructions from all the collected samples from cases and healthy controls. RNA quantification was done by using nanodrop by taking OD on 260/280, and further, 100 ng of total RNA was used to synthesize the cDNA using the manufacturer provided kit protocol (Verso, Thermo scientific, USA).
10
Trizol-based RNA Extraction and cDNA Synthesis
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The extraction of RNA was done through all untreated and treated cell lines using Trizol (Invitrogen) reagent as per the instruction given by manufacturers and stored at -80°C until additional necessary step for cDNA synthesis. RNA concentrations and purity were measured using a Thermo Scientific NanoDrop™ 2000 spectrophotometer. Hundred nanogram of total RNA, without reverse transcriptase and DNA controls, was used to synthesize cDNA using (Verso, Thermo scientific, USA) following the manufacturer's protocol.
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