Total RNA was isolated using the PeqGold Total RNA Isolation Kit (VWR International) according to the manufacturer’s instructions; 1 μg RNA was reverse transcribed using random hexamers (Fermentas) and murine leukemia virus reverse transcriptase (Thermo Scientific Fisher). Primers were designed using the software “Primer3,” and sequences are given in Supplementary Table S1 . Real-time PCR was done with the SsoAdvanced Universal SYBR Green Supermix (BioRad) using the StepOnePlus instrument (Applied Biosystems), and relative mRNA expression normalized to GAPDH. Fold changes in mRNA expression were calculated according to the 2-ΔΔCT method. Results are presented as mean fold induction of averaged Ct values of triplicates.
Peqgold total rna isolation kit
Manufactured by Avantor
Sourced in Germany, United States
The PeqGold Total RNA Isolation Kit is a laboratory equipment product designed for the purification of total RNA from various biological samples. It provides a simple and efficient method for extracting high-quality RNA for various downstream applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (4 mentions)
Murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Random hexamer, by Thermo Fisher Scientific (2 mentions)
Steponeplus instrument, by Thermo Fisher Scientific (2 mentions)
Stepone plus detector system, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Maxima rt, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Biopulverizer, by Biospec (1 mentions)
Quantitect rt kit, by Qiagen (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Pg lps, by InvivoGen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using peqgold total rna isolation kit
1
Total RNA Isolation and qPCR Analysis
2
Bulk 3'-end sequencing of RNA
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Total RNA was isolated from MC after four weeks of culture with the PeqGold Total RNA Isolation Kit (VWR, Radnor, PA, USA). Quality and integrity of total RNA was controlled using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies). To prepare a library of barcoded cDNA for bulk 30-sequencing, poly(A)-mRNA was reverse transcribed with Maxima RT polymerase (Thermo Fisher Scientific, Munich, Germany) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. The 5′ ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adaptor. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing, a TruSeq adapter was ligated and 3′-endfragments were finally amplified using primers with Illumina P5 and P7 overhangs. The library was sequenced on a NextSeq 500 (Illumina).
High-throughput gene expression data analysis was carried out using the R environment for statistical computing. Briefly, genes were normalized using DeSeq2 and a principal component analysis was performed. Next, pairwise comparisons were performed using edgeR and gene set enrichment analysis (GSEA). Additional gene set analysis was performed using the Degust webtool.
High-throughput gene expression data analysis was carried out using the R environment for statistical computing. Briefly, genes were normalized using DeSeq2 and a principal component analysis was performed. Next, pairwise comparisons were performed using edgeR and gene set enrichment analysis (GSEA). Additional gene set analysis was performed using the Degust webtool.
3
Quantifying Gene Expression in Osteoblasts and Osteoclasts
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Total RNA from primary osteoblasts or osteoclasts of four wild-type and Gnptgko mice each was isolated with the PEQ Gold Total RNA Isolation Kit (VWR) according to manufacturer’s instructions. For relative quantitative messenger RNA (mRNA) analysis, RNA isolation from cultured cells, complementary DNA (cDNA) synthesis, and quantitative real-time PCR using predesigned Taqman assays (Thermo Fisher Scientific) was performed as previously described [9 (link)]. The relative mRNA level of each analyzed gene was normalized to the level of Gapdh mRNA in the same cDNA sample using the comparative CT method (2–ΔΔCT).
4
Microarray Analysis of Total RNA from iBACs
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Total RNA from iBACs was isolated with PeqGOLD total RNA Isolation Kit (VWR, Darmstadt, Germany) according to manufacturer's recommendations. Two hundred nanograms of total RNA were prepared for Affymetrix hybridizations on Mouse Gene 2.0 ST Array. Raw array data were normalized using the RMA method implemented in CarmaWeb (https://carmaweb.genome.tugraz.at/carma/ ). For DAVID functional annotation, Ensembl Gene IDs of differentially regulated gene lists were submitted to the DAVID website [40 (link)]. KEGG pathways were considered significantly enriched if the Benjamini-Hochberg corrected p-value was < 0.05.
5
Quantifying E-Selectin Expression via qPCR
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Total RNA was isolated using the PeqGold Total RNA Isolation Kit (VWR International, Vienna, Austria), according to the manufacturer’s instructions. A total of 1 µg RNA was reverse transcribed using random hexamers (Fermentas) and murine leukemia virus reverse transcriptase (Thermo Scientific Fisher, Vienna, Austria). Primers were designed using the software “Primer3”. Following primer sequences were used for qPCR: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-AGAAGGCTGGGGCTCATTT-3′and reverse, 5′-CTAAGCAGTTGGTGGTGCAG-3′; E-Selectin (SELE) forward, 5′-CCTGTGAAGCTCCCACTGA-3′, and reverse 5′- GGCTTTTGGTAGCTTCCATCT-3′. Real-time PCR was done with the SsoAdvanced Universal SYBR Green Supermix (BioRad), using the StepOnePlus instrument (Applied Biosystems, Foster City, CA, USA), and relative mRNA expression was normalized to GAPDH. Fold changes in the mRNA expression were calculated according to the 2-ΔΔCT method. Observed effective concentration and the corresponding inhibition of three independent experiments are given as the percentage of maximum inhibition of the E-Selectin expression, compared to the untreated IL-1β control (Table 3 ).
6
Quantitative mRNA Expression Analysis
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Cellular RNA was isolated using the PeqGOLD Total RNA Isolation Kit (VWR, Darmstadt, Germany). RNA from tissue was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. Reverse transcription for cDNA generation was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). mRNA expression was assessed using real-time PCR using the StepOne Plus Detector system and SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was normalized to TfIIβ in cell lysates and 18s rRNA in tissues. Relative mRNA expression levels were calculated using averaged 2−ddCt values for each biological replicate [45 ]. For primer sequence, see Supplementary Table 1 .
7
Quantitative mRNA Expression Analysis
8
Total RNA Isolation and RT-qPCR Analysis
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Total RNA was isolated using the PeqGold Total RNA Isolation Kit (VWR International, Radnor, USA, #732-2868) according to the manufacturer’s instructions. A total of 1 μg RNA was reverse-transcribed using random hexamers (Fisher Scientific, Schwerte, Germany; #SO142) and murine leukemia virus reverse transcriptase (Fisher Scientific, #10338842). Primers were designed using the software “Primer3”, sequences are given in Table S2 (Supplementary Materials) . Real-time PCR was performed with the SsoAdvanced Universal SYBR Green Supermix (BioRad, Vienna, Austria, #1725272) using the StepOnePlus instrument (Applied Biosystems, Foster City, CA, USA), and relative mRNA expression normalized to GAPDH. Fold changes in mRNA expression were calculated according to the 2-ΔΔCt method. Results are shown as mean fold induction of averaged Ct-values of triplicates.
9
Quantifying mRNA Expression in HUVEC Cells
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Total RNA was isolated from HUVEC using the PeqGold Total RNA Isolation Kit (VWR International, Radnor, United States, #732-2868) according to the manufacturer’s recommendations. 1 μg RNA was reverse-transcribed using random hexamers (Fisher Scientific, Schwerte, Germany; #SO142) and murine leukemia virus reverse transcriptase (Fisher Scientific, #10338842). Primers were designed using the software “Primer3,” and sequences given in Supplementary Table S2 . qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (BioRad, Vienna, Austria, #1725272) in a StepOnePlus real-time therocycler (Applied Biosystems, Foster City, CA, United States). Relative mRNA expression was normalized to GAPDH. Triplicate samples were analyzed except for Figure 1A where duplicates were performed. Fold changes in mRNA expression were calculated according to the 2-ΔΔCt method (Livak and Schmittgen, 2001 (link)).
10
Microarray Analysis of Total RNA from iBACs
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