Overnight stationary cells were inoculated into 3 ml LB + Sm medium, grown at 37°C until mid-late exponential phase (OD 600 0.5–0.8), harvested and total RNA extracted with TRIzol reagent (Life Technologies). RNA was treated with Turbo DNase I for 30 min (Life Technologies) and subjected to qRT-PCR as previously described [71 (link)]. Briefly, 1 μg total RNA was used for the reverse transcription reaction with Superscript III first strand synthesis system with random hexamers (Life Technologies). The synthesized cDNA was subjected to real time-PCR amplification using the Fast SYBR Green Master Mix kit (Life Technologies) on the StepOnePlus platform (Life Technologies) using primers shown in S6 Table . The amplification data was analyzed by ΔΔCT method utilizing rpoC mRNA as internal control.
Steponeplus platform
Manufactured by Thermo Fisher Scientific
Sourced in United States
The StepOnePlus platform is a real-time PCR system designed for a wide range of applications in molecular biology research. It provides accurate, reliable, and efficient performance for real-time quantitative PCR (qPCR) analysis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Mirneasy mini kit, by Qiagen (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
Tb green premix ex taq 2 kit, by Takara Bio (4 mentions)
Tri reagent, by Merck Group (3 mentions)
Superscript 2, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Nd 100 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Turbo dnase 1, by Thermo Fisher Scientific (2 mentions)
Fast sybr green master mix kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system with random hexamers, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Purelink mini kit, by Thermo Fisher Scientific (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq dye, by Takara Bio (2 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (2 mentions)
46 protocols using steponeplus platform
1
Quantitative RT-PCR for Gene Expression
2
Quantifying Human-Chimpanzee Transcriptomic Differences
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Primers for qPCR and amplicon sequencing are listed in Supplementary Table 1 . RNA from fibroblasts and iPSCs were reverse transcribed by random hexamers using Superscript III and the resulting first cDNA were used as templates for qPCR. qPCR were performed under standard two-step cycling conditions using TaqMan Universal PCR master mix (Life Technologies) on StepOne Plus platform (Life Technologies). One tenth of the double strand cDNA from the TruSeq procedure for library preparation were saved and used as templates for amplicon sequencing. Since the templates for amplicon sequencing were obtained from the RNA-seq procedures after spike-in of chimpanzee RNA, they represent both human and chimpanzee transcripts. Amplicons were prepared by standard two-step PCR using SYBR Premix ExTaq (Takara). Purified amplicons were subjected to library preparation by TruSeq DNA Sample Preparation Kit (Illumina). Amplicon sequencing was performed on MiSeq (Illumina) platform with a paired-end read length of 250-bp. Human and chimpanzee reads were discriminated based on SNVs in the amplicon and normalized gene expression levels were calculated by dividing the human reads with the chimpanzee reads.
3
Quantitative Analysis of RNA Expression
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Cells from overnight (stationary-phase) cultures were inoculated in triplicate into 5 ml LB or M9 and grown at 37°C until exponential phase (OD600 of ∼0.3) or stationary phase. Total RNA was extracted from harvested cells with TRIzol reagent (Life Technologies). RNA was treated with Turbo DNase I for 30 min (Life Technologies) and subjected to quantitative reverse transcriptase PCR (qRT-PCR) as previously described (38 (link)). Briefly, 1 μg total RNA was used for the reverse transcription reaction with Superscript III first strand synthesis system with random hexamers (Life Technologies). Real-time PCR amplification of the synthesized cDNA was conducted using the Fast SYBR green Master Mix kit (Life Technologies). Each of the three biological replicates was analyzed in technical triplicate on the StepOnePlus platform (Life Technologies) using primers shown in Table S3 . The data were analyzed by the ΔΔCT method using rpoC mRNA as an internal control. Log2 fold change was calculated from the ΔΔCT results.
4
Quantifying ABCG2 mRNA Expression
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mRNA samples from cells expressing RUSH-ABCG2 were isolated and purified 24 h after transfection using a PureLink MiniKit (Thermo Fisher Scientific, cat. 12183018A) according to the manufacturer’s instructions. After cDNA conversion (Thermo Fisher Scientific, High-capacity cDNA reverse transcription kit, cat. 4368814), PCR reactions were run on a StepOnePlusTM platform (Thermo Fisher Scientific) using qPCR probes for ABCG2 (Thermo Fisher Scientific, cat. 00184979) and RPLP0 (Thermo Fisher Scientific, cat. 99999902). ABCG2 mRNA levels were normalized first to RPLP0 and then to ABCG2-wt mRNA levels using the ΔΔCt method.
5
Transcriptional Profiling of Dot1l Mutant Endothelial Cells
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Dot1l cKO ECs from E15.5 skins and cultured Dot1l overexpression ECs were used for RT-qPCR. Total RNAs were extracted using RNeasy Plus Mini Kit (Qiagen). SMARTer Pico PCR cDNA synthesis kit (Takara) along with Advantage 2 PCR Kit (Takara) and TOPscript RT DryMix cDNA synthesis kit (Enzynomics) were used for cDNA synthesis of Dot1l cKO ECs and Dot1l overexpression ECs, respectively. RT-qPCR was performed on StepOnePlusTM platform (Thermo Fisher Scientific) using Fast SYBR® Green Master Mix (Thermo Fisher Scientific). Gene expression in each sample was normalized to the expression of Gapdh. The sequences for primers used for RT-qPCR are as follows: Gapdh; 5′-CATGGCCTTCCGTGTTCCTA-3′, 5′-GCCTGCTTCACCACCTTCTT-3′, Dot1l; 5′-TGGCAAGCCTGTCTCCTACT-3′, 5′-CTGCTCCTCCCTGAGTTTTG-3′, Mmp3: 5′- ACATGGAGACTTTGTCCCTTTTG-3′, 5′-TTGGCTGAGTGGTAGAGTCCC-3′ and Cldn1 5′-GGGGACAACATCGTGACCG-3′, 5′-AGGAGTCGAAGACTTTGCACT-3′.
6
Quantitative Real-Time PCR Gene Expression
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Total RNA was extracted using TRI Reagent (Sigma #T9424) or by Renozol (Genoplast #BMGPB1100–2) followed by Total RNA Mini column purification kit (A&A Biotechnology #031–100). 2 μg of RNA was subjected to reverse transcription using M-MLV enzyme (Promega #M1705), dNTP mix 100 mM each (BLIRT #RP65) and oligo (dT)18 primers (Bioline #BIO-38029). The RT-qPCR reaction was performed using SensiFAST SYBR Hi-ROX Kit (Bioline #BIO-92020) on the StepOnePlus™ platform (Thermo Fisher Scientific) according to MIQE guideline. Primers sequences are listed in the Supplementary Information. The comparative 2-ΔΔCt method was used to determine the relative mRNA level using StepOnePlus software. 18SrRNA was used as a reference control. Data are presented as mean values ± SD; n = 3–5. Statistical significance was assessed using unpaired Student’s t-test with Welch’s correction and p ≤ 0,05 was estimated as significant (*p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001; ****p ≤ 0.0005).
7
Quantitative RT-PCR of IL-7-Cultured B Cells
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For qRT-PCR on IL-7-cultured B cell precursors, cells were typically harvested on day 7 after transduction, dead/apoptotic cell-depleted, and snap-frozen on dry ice. For thymus and BM, cells were isolated freshly from 53BP1−/− mice and snap-frozen on dry ice. Stimulated B cells were obtained from the spleen and cultured as described previously (Feldhahn et al., 2012 (link)). RNA was isolated as for RNA-seq, and cDNA was generated using the RevertAid cDNA synthesis kit (Thermo Fisher Scientific). qPCR was performed using a SYBR green master mix (Sigma JumpStart) on a StepOnePlus platform (Thermo Fisher Scientific). Information of primers, PCR conditions, and analysis can be found in the Supplemental Experimental Procedures . Analysis of the IGH locus for genomic loss was done as by Jankovic et al. (2013) (link) using the primers and conditions described.
8
MiRNA and piRNA Expression Quantification
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cDNA synthesis was performed using the miRCURY LNA RT Kit (QIAgen), with 0.56 μl of RNA templates added to each reaction according to the equation “Template RNA [µl] = Elution volume [µl]/Original sample volume [µl] * 8 [µl]” (https://www.qiagen.com/us/resources/resourcedetail?id=34039664-5bf4-42b1-9858-f4c28dace788&lang=en ). qPCR was carried out using the miRCURY Probe PCR Kit, run on a StepOnePlus™ platform (Thermo Fisher Scientific) according to the manufacturer’s instructions. miRCURY LNA miRNA Probe Assays were applied in the case of hsa-miR-122-5p, while custom-made miRCURY LNA miRNA Custom Probe Assays were used in the case of hsa_piR_016658 (Table 1
9
Quantifying ABCG2 mRNA Expression
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mRNA was purified from the stable, ABCG2-expressing HeLa cells with PureLink MiniKit (Thermo Fisher Scientific, cat. 12183018A) according to the manufacturer’s instructions. After cDNA conversion (Thermo Fisher Scientific, High capacity cDNA reverse transcription kit, cat. 4368814) mRNA levels were determined with qPCR probes against ABCG2 (Thermo Fisher Scientific, cat. 00184979) and RPLP0 (cat. 99999902), with TaqMan Universal Master Mix (Thermo Fisher Scientific, cat. 4304437) according to the manufacturer’s protocol. Reactions were run on a StepOnePlus™ platform (Thermo Fisher Scientific), and ABCG2 mRNA levels were normalized with ΔΔCt method to RPLP0 mRNA levels.
10
miRNA Expression Analysis Using miRCURY LNA
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The expression analysis was performed using the miRCURY LNA™ Universal RT miRNA PCR Assay (Qiagen) according to the manufacturer's instructions. Briefly, RNA samples (5 ng/µl) were reverse-transcribed using the miRCURY LNA™ RT Kit. UniSp6 RNA spike-in template was added to each reaction for cDNA synthesis control. cDNAs of placental and cell culture RNAs were 1:80 diluted, whilst templates from exosomal and Agobound miR fractions were 1:10 diluted before subsequent amplification. RT-PCR was carried out using miRCURY SYBR® Green master mix with specific LNA™ PCR primer sets, run on a StepOnePlus™ platform (Thermo Fisher Scientific) using the manufacturer's instructions. The relative expression of the investigated miRNAs was calculated based on the CT method and was normalized to hsa-miR-103a internal control miRNA. The miRBase identifiers and seed sequences of investigated and reference miRNAs are listed in Table 1.
Melting curve analysis was carried out following each PCR run for evaluating the specificity of the assays.
Melting curve analysis was carried out following each PCR run for evaluating the specificity of the assays.
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