Sort plates were thawed on ice and briefly centrifuged before adding 11.7 µl per well of ice-cold RT-PCR master mix containing 5.8 µl nuclease free water, 4.8 µl 5× FS buffer (Life Technologies), 1 µl 25 mM dNTP mix (Roche Applied Science, Indianapolis, Indiana), and 0.1 µl random primers (Life Technologies, catalog no. 48190-011) per well. Plates were again briefly centrifuged, incubated at 65°C for 5 minutes, and returned to ice. Then 8.3 µl of ice-cold RT-PCR master mix (2) containing 4.8 µl nuclease-free water, 0.8 µl 5× FS buffer, 0.2 µl RNasin, 2 µl 0.1M dTT, and 0.5 µl SuperScript III RT (Life Technologies) were added per well, and cDNA was generated with the following PCR program: 1 cycle for 5 minutes at 25°C, 1 cycle for 60 minutes at 50°C, 1 cycle for 15 minutes at 70°C, and 4°C forever. cDNA was stored at 4°C (short-term) or −20°C (long-term).
Fs buffer
Manufactured by Thermo Fisher Scientific
5× FS buffer is a concentrated solution used to prepare samples for analysis in flow cytometry applications. It is designed to maintain the integrity and stability of cells or particles during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3, by Thermo Fisher Scientific (6 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (5 mentions)
Dntps, by Thermo Fisher Scientific (5 mentions)
Rnaseout, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Random primer, by Thermo Fisher Scientific (3 mentions)
Random primers, by Merck Group (2 mentions)
Stainless steel bead, by Qiagen (2 mentions)
Fibrous tissue mini kit, by Qiagen (2 mentions)
Chloroform, by Merck Group (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Tissuelyser lt, by Qiagen (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
M mlv transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Agilent Technologies (1 mentions)
Fla 7000 imaging analyzer, by Fujifilm (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
15 protocols using fs buffer
1
Reverse Transcription PCR Protocol
2
Quantifying Gene Expression in Immune Cells
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2.5 µg total RNA of each spleen was subjected to a reverse transcription reaction in a total volume of 40 µl employing 5 × FS-Buffer, 3 µg random hexamer primer, 200 U M-MLV transcriptase, 0.02 µmol dNTPs, and 0.4 µmol DTT (all InvitrogenTM GmbH) as well as 40 U RNAse inhibitor (Agilent Technologies). 70 °C denaturation step was followed by a 1.5 h 42 °C reverse transcription step. 1 µl of individual cDNA preparations from each IL-10R-blocked and isotype-treated mouse, respectively, was pooled. The two pools were diluted 1:200 for subsequent qPCR which was performed as described previously47 (link). Transcripts of three housekeeping genes and 32 genes involved in cytokine-, interferon-, chemokine- and innate immunity-related signaling were quantified as fold changes using the ΔΔCT-method. For a detailed list of genes and primer sequences, see supplemental Table S2 . Transcripts reaching a threshold fold change of 1.5 were regarded as potentially regulated and subjected to RT-qPCR in non-pooled samples.
3
Quantifying τm^5U in mt tRNA^Leu(UUR)
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To quantify τm5U frequency in mt tRNALeu(UUR), we employed a primer extension method assisted by chemical derivatization of τm5U (31 (link)). The total tRNA fraction was purified by electrophoresis on a 10% polyacrylamide gel containing 7 M urea and gel extraction. Then, CMC [N-cyclohexyl-N’-β-(4-methylmorpholinium) ethylcarbodiimide] derivatization of mt tRNALeu(UUR) in the total RNA fraction was performed as described previously (6 (link),32 (link)). The CMC-derivatized mt tRNALeu(UUR) was subjected to a reverse-transcription reaction with 5′-[32P]-labeled primer for mt tRNALeu(UUR) (5′-ACCTCTGACTGTAAAG-3′) and SuperScript III Reverse Transcriptase (Invitrogen) in buffer containing 1 × FS buffer (Invitrogen), 3.75 mM MgCl2, 0.0375 mM each of dATP and dTTP, and 0.075 mM ddGTP at 55°C for 1 h. The cDNAs were subjected to 20% PAGE with 7 M urea (20 × 20 cm2, 0.35 mm). The gel was exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm).
4
Avidin-Biotin Cell Seeding Optimization
5
Single-Cell Reverse Transcription Protocol
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Reverse transcription was performed in an Eppendorf vapo.protect thermal cycler. Nine microliters of reverse transcription buffer 1 containing 5 μl 2x FS buffer (Invitrogen), 3 μl of lysis buffer (5% Nonidet P-40/Igepal CA630, SIGMA) and 1 μl of random primers (150 ng/μl, Invitrogen) were added to each well containing a sorted cell. The first reverse transcription step was performed at 65°C for 10 min followed by 25°C for 3 min. The lysed cells were kept on ice for at least 1 min before pipetting 5.5 μl of the second mastermix containing 0.5 μl of 100 U SSIII reverse transcriptase (Invitrogen), 2 μl of 5x FS buffer (Invitrogen), 2 μl of 0.1M DTT (Invitrogen) and 1 μl of 2.5 mM dNTPs (Invitrogen). After mixing and a short spin, the plates were incubated at 37°C for 1 h followed by 70°C for 15 min to inactivate the enzyme.
6
RNA Extraction, cDNA Synthesis, and Gel Quantification
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After extraction, total RNA was subjected to DNAse treatment (TURBO DNAse, Invitrogen). First strand (FS) synthesis was performed using a 5X FS master mix composed of 0.5 μL 1X FS buffer (Invitrogen), 0.5 μL random primer mix, 5 μg RNA, and up to 7 μL of water. This mixture was heated to 95°C for 1 minute, followed by 65°C for 1 minute, then at room temperature for 1–2 minutes. All 7 μL were added to 5 μL of 1X reverse transcriptase (RTase) master mix. 1X RTase master mix included 1.5 μL 5X FS buffer, 1 μL 0.1 M dithiothreitol (DTT, Invitrogen), 1 μL 10 mM dNTPs (Thermo Scientific), 0.5 μL 40 U/μL RNAse inhibitor (RNAsIN, Promega), and 0.5 μL 200 U/μL SuperScript III RTase (Invitrogen) or water for no RTase control. The final 12 μL mixture was incubated at room temperature for 5 minutes followed by 48°C for 25 minutes. The Zymo Research DNA Clean & Concentrator-5 kit was used to purify single-stranded DNA (ssDNA). PCR was performed on ssDNA, and products were run on a 2% agarose gel. Bands were quantified from unsaturated tif files using ImageJ (NIH), and data was visualized using GraphPad Prism (v9.4.0 for macOS).
7
RNA Extraction and cDNA Synthesis Protocol
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Tissue was quickly dissected and frozen on either dry ice or liquid nitrogen and stored at −80 °C. Tissue was homogenized in a Trizol reagent (QIAzol Lysis Reagent, Qiagen) using a stainless steel bead (Qiagen) and a TissueLyser LT (Qiagen) for 3 min at 20 Hz. Then, 200 μl chloroform (Sigma-Aldrich) was added and tubes were shaken vigorously for 15 sec and left at RT for 2 min, followed by centrifugation at 4 °C for 15 min at 12,000×g. The aqueous phase was mixed 1:1 with 70% ethanol and further processed using RNeasy Lipid Mini Kit following the instructions provided by the manufacturer. For muscle tissue, the lysis procedure, described the enclosed protocol in the Fibrous Tissue Mini Kit (Qiagen), was followed. After RNA extraction, RNA content was measured using a NanoDrop 2000 (Thermo Fisher) and 500 ng of RNA was converted into cDNA by mixing FS buffer and DTT (Thermo Fisher) with Random Primers (Sigma-Aldrich) and incubated for 3 min at 70 °C followed by addition of dNTPs, RNase out, Superscript III (Thermo Fisher) and placed in a thermal cycler for 5 min at 25 °C, 60 min at 50 °C, 15 min at 70 °C, and kept at −20 °C until further processing.
8
Reverse Transcription of RNA to cDNA
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A total of 2 μg
of RNA extracted from each sample is reverse-transcribed into cDNA
using M-MLV reverse transcriptase (Thermo Fisher Scientific, Waltham,
MA, USA). The reaction mixture containing 5× FS buffer (Thermo
Fisher Scientific), 0.1 M DTT (Invitrogen), M-MLV, nuclease-free water
(Fermentas), and RNase inhibitor (Thermo Fisher Scientific) were added
in the PCR tubes in specified quantities.
In each tube, 9 μL
of the RNA sample, 2 μL of dNTPS, and 1 μL of the reverse
primer of TRIM14 were added. The primer sequences for TRIM11, TRIM14,
and TRIM25, as well as for GAPDH as a control, are shown inTable 1 . GAPDH cDNA synthesis
and subsequent quantification were performed and used as a positive
control for normalization. Each tube was short-spanned and incubated
in a thermocycler for 1 h at 42 °C and 5 min at 85 °C. The
cDNA quantification was carried out by a Nano-Drop (ND-100 UV/vis,
USA).
of RNA extracted from each sample is reverse-transcribed into cDNA
using M-MLV reverse transcriptase (Thermo Fisher Scientific, Waltham,
MA, USA). The reaction mixture containing 5× FS buffer (Thermo
Fisher Scientific), 0.1 M DTT (Invitrogen), M-MLV, nuclease-free water
(Fermentas), and RNase inhibitor (Thermo Fisher Scientific) were added
in the PCR tubes in specified quantities.
In each tube, 9 μL
of the RNA sample, 2 μL of dNTPS, and 1 μL of the reverse
primer of TRIM14 were added. The primer sequences for TRIM11, TRIM14,
and TRIM25, as well as for GAPDH as a control, are shown in
and subsequent quantification were performed and used as a positive
control for normalization. Each tube was short-spanned and incubated
in a thermocycler for 1 h at 42 °C and 5 min at 85 °C. The
cDNA quantification was carried out by a Nano-Drop (ND-100 UV/vis,
USA).
9
RNA Extraction and cDNA Synthesis from Skeletal Muscle and BAT
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Tissues (skeletal muscle and BAT) were quickly dissected and frozen either on dry ice or with liquid nitrogen and stored at −80°C. Tissues were homogenized in a TRIzol reagent (QIAzol Lysis Reagent, Qiagen) using a stainless steel bead (Qiagen) and a TissueLyser LT (Qiagen) for 3 minutes at 20 Hz. Then, 200 μL of chloroform (Sigma‐Aldrich) was added and tubes were shaken vigorously for 15 seconds and left at RT for 2 minutes, followed by centrifugation at 4°C for 15 minutes at 12 000 g. The aqueous phase was mixed 1:1 with 70% ethanol and further processed using RNeasy Lipid Tissue Mini Kit (Qiagen) following the instructions provided by the manufacturer. For muscle tissue, the lysis procedure described in the enclosed protocol in the Fibrous Tissue Mini Kit (Qiagen) was followed. After RNA extraction, RNA concentration and purity were measured using a NanoDrop 2000 (Thermo Fisher). A total of 500 ng of RNA (1000 ng RNA in the case of BAT) was converted into cDNA by mixing FS buffer and DTT (Thermo Fisher) with Random Primers (Sigma‐Aldrich) and incubated for 3 minutes at 70°C. This was followed by the addition of dNTPs, RNase out, Superscript III (Thermo Fisher), and cDNA was synthesized in a thermal cycler using following steps: 5 minutes at 25°C, 60 minutes at 50°C, 15 minutes at 70°C. cDNA was diluted 1:100 in nuclease‐free water and kept at −20°C until further processing.
10
cDNA Synthesis from Mouse and Human RNA
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cDNA was synthesized from 500 ng mouse organ RNA, 100 ng human and murine neutrophil RNA or 250 ng THP-1 cell RNA using the Super-Script III kit (Thermo Fisher #18080044). RNA was mixed with sterile water (Amgros #744128) and 5x FS Buffer (Thermo Fisher #18080044), random primers at 3.2 μg/μL (Roche #11034731001), and 0.1 M dithiothreitol (Thermo Fisher #18080044). This was heated to 70°C for 3 min in an Eppendorf Vapo.protect Mastercycler® Pro (Eppendorf) and subsequently cooled on ice. Next, 10 mM dNTPs (Thermo Fisher #10297018), RNaseOUT (40 U/μL; Thermo Fisher #10777019) and Super-Script III (200U/μL; Thermo Fisher #18080044) were added, and the cDNA synthesis was run with the following program: 25°C for 5 min, 50°C for 60 min, 70°C for 15 min, followed by cooling to 4°C. A negative control sample with no RNA was prepared with all experiments. The cDNA was stored at −80°C.
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