For tuft cell qPCR, CD45loRFP+EpCAM+ and CD45loRFP−EPCAM+ populations from small intestinal epithelium of Il25Flare25/Flare25 mice were sorted into Buffer RLT (Qiagen) using an Aria (BD Biosciences). To validate Gnat3−/− mice, CD45loEpCAM+ epithelial cells from the small intestine were sorted into Buffer RLT (Qiagen). RNA was isolated using the Micro Plus RNeasy kit (Qiagen) and reverse transcribed using SuperScript Vilo Master Mix (Life Technologies). For tritrichomonas quantification, total DNA was isolated from cecal contents using the QIAmp Fast DNA Stool Mini Kit (Qiagen). Cecal DNA or cDNA were used as template for quantitative PCR with Power SYBR Green reagent on a StepOnePlus cycler (Applied Biosystems). For mouse cells, transcripts were normalized to Rps17 (40S ribosomal protein S17) expression. For cecal DNA, transcripts were normalized to bacterial 16s rRNA.
Rlt buffer
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, Netherlands, France, Switzerland, Spain, Canada, Japan, Denmark, Sweden, China
RLT buffer is a specialized lysis buffer used for the extraction and purification of RNA from a variety of sample types. It is designed to efficiently lyse cells and inactivate RNases, thereby preserving the integrity of the extracted RNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (447 mentions)
Rneasy micro kit, by Qiagen (124 mentions)
Rneasy kit, by Qiagen (114 mentions)
β mercaptoethanol, by Merck Group (84 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (80 mentions)
Rneasy plus mini kit, by Qiagen (54 mentions)
Qiashredder, by Qiagen (52 mentions)
Nanodrop, by Thermo Fisher Scientific (48 mentions)
Iscript cdna synthesis kit, by Bio-Rad (44 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (42 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (42 mentions)
Qiashredder column, by Qiagen (37 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (36 mentions)
Rneasy plus micro kit, by Qiagen (33 mentions)
Trizol, by Thermo Fisher Scientific (33 mentions)
2 mercaptoethanol, by Merck Group (33 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (30 mentions)
2100 bioanalyzer, by Agilent Technologies (28 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (28 mentions)
Rnalater, by Thermo Fisher Scientific (27 mentions)
1 835 protocols using rlt buffer
1
Tuft Cell and Epithelial Cell qPCR
2
Adipose Tissue Fractionation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Epididymal adipose tissues were removed at the time of sacrifice and minced finely into small pieces and resuspended in 5 ml Hanks’ Balanced Salt Solution (HBSS, H9269, Sigma), 0.1 g bovine serum albumin (BSA, A8806, Sigma), and 10 mg collagenase type II (C6885, Sigma). The tissue was completely disaggregated by incubation in a 37 °C shaker for approximately 15 min. The digested material was topped with 5 ml of ice-cold MACS buffer (2 mM EDTA, 0.5% BSA in PBS) and allowed to settle for 5 min at room temperature. The adipocyte fraction floating on top was collected by pipetting, washed twice with MACS buffer and then snap frozen in Buffer RLT (Qiagen) for RNA extraction. The remainder of the digested solution (approximately 9 ml) was filtered through a 100 μm nylon mesh cell strainer (Falcon 352360) and centrifuged at 400 × g for 5 min. The pellet containing the stromalvascular fraction (SVF) was collected and washed once with MACS buffer and then resuspended with CD11b microbeads (130-049-601-Millteyni Biotec) in MACS buffer. The CD11b positive and negative cells (SVF) were separated and collected using MACS LS columns (130-042-401, Miltenyi Biotec) that were placed onto a magnetic field of a MACS separator (Miltenyi Biotec). The cells were centrifuged at 400 × g for 5 min and snap frozen in Buffer RLT (Qiagen) for RNA extraction.
3
Validating Gal-9 Induced B Cell Transcriptome
4
Mouse and Human Keratinocyte IFN Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary mouse keratinocytes in 12-well plates were grown to confluence for 3–4 days after isolation. Recombinant murine IFN-β (5 ng/mL; PBL #12401) or IFN-λ3 (5 ng/mL; PBL# 12820) was resuspended in fresh keratinocyte growth media, and 1 mL of IFN suspension or media alone was added to cells. Twenty-four hours later, the supernatant was removed, 350 µL of Buffer RLT (Qiagen) was added to each well, and samples were stored at −80°C until RNA extraction. A549 cells were treated similarly at confluence in 6-well plates with recombinant human IFN-β (5 ng/mL; PBL #11415) or IFN-λ2 (50 ng/mL; PBL #11720) or media only, resuspended in A549 growth media. Then, 8 hours later, the supernatant was removed, 350 µL of Buffer RLT (Qiagen) was added to each well, and samples were stored at −80°C until RNA extraction.
5
Investigating Mycobacterium Tuberculosis Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We first analyze the systemic effect of the disease on the basal profiles of MoTB gene expression compared to MoCT (Supplementary Figure 1 ). For this, adherent cells were lysed in buffer RLT (Qiagen) with 1% β-mercaptoethanol and stored at −80°C until use for RNA extraction. Second, we analyzed the effect of in vitro infection with the clinical isolates of Mtb UT127 and UT205 in MoTB and MoCT (Supplementary Figure 1 ). Monocytes were infected for 6 h with Mtb at a multiplicity of infection (MOI) of 10:1. The cells were extensively washed with pre-warmed (37°C) DPBS supplemented with 0.5% AB+ inactivated human serum to eliminate non-ingested bacteria. Then, adherent cell were lysed in buffer RLT (Qiagen) with 1% β-mercaptoethanol and stored at −80°C until use for RNA extraction. Three experimental settings were evaluated, including (1) In vitro non-infected samples (NI) used as a control, (2) samples in vitro infected with Mtb UT127, and (3) samples in vitro infected with Mtb UT205 (Supplementary Figure 1 ). The percentage of infected monocytes in the experimental conditions used in this study (MOI 10:1) was defined in preliminary experiments using the Kinyoun staining. At least 400 cells in randomly selected fields were counted. In all cases, the proportion of infected macrophages was superior to 85% (data not shown).
6
Retina RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Retinae were dissected immediately after CO 2 inhalation and snap frozen in Buffer RLT (Qiagen, UK) containing β-Mercaptoethanol (10 µL/mL Buffer RLT) as recommended by the manufacturer. Total RNA was extracted using Qiagen's RNeasy Mini kit (Qiagen, UK) and reverse transcribed (300 ng of RNA per sample) into cDNA using a Superscript II Reverse Transcriptase kit (Invitrogen, UK) as per manufacturer's instructions. cDNA samples were stored at -20°C until further use. Quantitative PCR was carried out in a total reaction volume of 10 µL containing 2 µL of cDNA (prior diluted 1:10 in water), 5 µl of 2× SYBR green (Fermentas, Canada), 0.5 µl of 10 mM forward and reverse primer and 2.5 µL RNase/DNase free water. Reactions were performed in triplicate using the LightCycler 480 Real-Time PCR System (Roche Diagnostics Ltd, West Sussex, UK). Expression of target genes relative to reference genes was calculated using the advanced relative quantification analysis module of the LightCycler 480 software 1.5 (Roche Diagnostics Ltd) based on the delta-delta CT method. Four housekeeping genes were tested (β-actin, 18S, RPLP0 and SDHA). Relative expression levels (relative to RPLP0 as this was the most stable housekeeping gene across samples) are presented in graphs. Primer sequences are shown in Table 1.
7
IFN Stimulation and Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SSP-9 cells were seeded in 24-well plates with 1.2 x 10 5 cells/well and grown overnight in L-15+ (8% FBS). Cells were stimulated in triplicate with 200 U/ml of recombinant IFNa1, IFNb, and IFNc (Svingerud et al., 2012) in L-15+ (8% FBS). Cells were harvested in RLT buffer (Qiagen) 12, 24, and 72 h post-stimulation. CHSE-GFP-SOCS1-blast and CHSE-GFP-blast cells were seeded in 24-well plates (1.5 x 10 5 cells/well) and grown overnight in L-15+ supplemented with 8% FBS and 15 μg/ml blasticidin. Cells were stimulated in triplicate with 500 U/ml of recombinant IFNa1 in L-15+ (8% FBS). Cells were harvested in RLT buffer (Quiagen) 24 h post-stimulation. RNA isolation, cDNA synthesis, and quantitative PCR were performed as described in Sections 2.10 and 2.11.
8
Dual RNA Isolation from Host-Fungus Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We designed an RNA isolation protocol for isolation of both fungal and murine RNA from the same sample based on step-wise isolation of host and fungal RNA (Suppl. Fig. 5 ). First, organs were aseptically homogenized in RLT buffer (Qiagen) with 1% β-Mercaptoethanol (β-ME) on ice water using an Ika T10 basic UltraTurrax homogenizer. Then, homogenates were centrifuged at 3,000 g at 4 °C for 3 min. The supernatant was used for mouse tissue RNA isolation with the RNeasy Mini Kit (Qiagen) as described by the manufacturer. The remaining pellets were vortexed on a mini-beadbeater (Precellys) for 5 sec at 5000 m/s in 1 ml RLT buffer (Qiagen) with 1% β-ME. After centrifugation at 4 °C, fungal RNA was isolated from the remaining pellets as previously described24 (link). The RNA quality was determined using a Bioanalyzer (Agilent Inc.), the quantity was measured with a Nanodrop ND1000 (Peqlab).
9
Tissue Homogenization and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 250 mg of frozen tissue from each CRC patient sample was ground in RLT buffer (Qiagen) using a tissue homogenizer then purified following the RNeasy Mini Kit (Qiagen) protocol. For cancer cell lines, RLT buffer was directly added to plated cells and the lysate passed through a QIAShredder (Qiagen) before continuing with the RNeasy Mini Kit protocol.
10
FACS-sorted population gene expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FACS-sorted populations were stimulated with PMA/Ionomycin (10 ng/mL) for 4 h at 37 °C. PBMCs were pellet down at 1500 rpm at 4 °C. 6249–10,000 cells in 5uL of RLT buffer (Qiagen) were hybridized with probes from the nCounter Human Inflammation v1 panel and 10,000 cells (except for 3 samples with ~5500–7700 cells) in 5uL of RLT buffer (Qiagen) were hybridized with probes from the nCounter Human Senescence custom panel at 65 °C for 19 h according to nCounter™ Gene Expression Assay Manual. The nCounter™ Digital Analyzer (GEN1) was used to quantify target molecules present in each sample. A high-density scan (600 fields of view) was performed.
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!