Total RNA from the terminal ileum, transverse colon, and mesenteric lymph nodes was isolated and transcribed as described elsewhere [57 (link)]. Approximately 10 mg of the tissue stored in RNAlater (Qiagen, Hilden, Germany) at −20 °C were moved to RLT buffer of the RNeasy Plus Mini kit (Qiagen) with zirconia beads (BioSpec Products, Bartlesville, OK). The tissue was homogenized in TissueLyser LT beadbeater (Qiagen). The next steps followed the manufacturer’s instructions. Five hundred ng of the total RNA (A260/A280 ≥ 2.0 as measured in 10 mM Tris-HCl buffer pH 7.5) were reverse transcribed by QuantiTect Reverse Transcription kit (Qiagen) according to manufacturer’s instructions. 20 μL of the synthesized cDNA was 1/10 diluted by PCR quality water (Life Technologies, Carlsbad, CA), and these PCR templates were stored at −25°C until the following real-time PCR.
Tissuelyser lt beadbeater
Manufactured by Qiagen
Sourced in United States, Germany
The TissueLyser LT is a beadbeater designed for efficient disruption and homogenization of biological samples. It uses high-speed shaking motion to agitate samples in the presence of metal or ceramic beads, effectively breaking down tissues and cells to release their contents for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect reverse transcription kit, by Qiagen (6 mentions)
2 mm zirconia beads, by Biospec (4 mentions)
Rneasy plus mini kit, by Qiagen (4 mentions)
Pcr quality water, by Thermo Fisher Scientific (3 mentions)
Rnalater, by Qiagen (2 mentions)
Zirconia beads, by Biospec (2 mentions)
Macconkey agar, by Merck Group (2 mentions)
Rnaeasy plus micro kit, by Qiagen (2 mentions)
Pcr grade water, by Thermo Fisher Scientific (2 mentions)
Brilliant green agar, by Thermo Fisher Scientific (1 mentions)
Dulbecco s pbs dpbs, by Thermo Fisher Scientific (1 mentions)
Quant it ribogreen rna assay kit, by Thermo Fisher Scientific (1 mentions)
Infinite m200, by Tecan (1 mentions)
Soya peptone, by Thermo Fisher Scientific (1 mentions)
Mupirocin, by Merck Group (1 mentions)
Anaerogen sachet, by Thermo Fisher Scientific (1 mentions)
Wilkins chalgren agar, by Thermo Fisher Scientific (1 mentions)
L cysteine, by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Rnalater, by Merck Group (1 mentions)
8 protocols using tissuelyser lt beadbeater
1
Isolation and Transcription of Total RNA
2
Quantifying Intestinal and Blood Microbiome
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Samples of peripheral blood were cultivated log 10 and diluted by PBS. Jejunum (40 cm of the proximal part of the jejunum) and ileum (40 cm segment of a terminal part of the small intestine containing the ileum and part of the distal jejunum) lavages were cut off, filled with 2 mL of Dulbecco’s PBS (DPBS; Life Technologies, Carlsbad, CA), gently kneaded, and rinsed. Colon lavage was obtained by placing the whole colon in a 90 mm Petri dish, cut into small pieces in 4 mL of DPBS. All lavages were vigorously vortexed. Further, 0.2 g of mesenteric lymph nodes, liver, and spleen were homogenized in 0.8 mL deionized water in a 2 mL Eppendorf tube containing two 3.2 mm stainless-steel beads in a TissueLyser LT beadbeater (Qiagen, Hilden, Germany) shaken for 3 min at 50 Hz. The intestinal lavages, tissue homogenates, and blood were serially diluted in PBS and cultivated in 90 mm Petri dishes with MRS agar for lactobacilli (Oxoid), MacConkey agar (Merck, Darmstadt, Germany) for E. coli or Brilliant green agar (Oxoid) for S. Typhimurium. The plates were incubated aerobically at 37 °C for 48 h for lactobacilli or 24 h for E. coli or S. Typhimurium. The CFU were counted from dishes optimally containing 20–200 colonies.
3
RNA Isolation and cDNA Synthesis for Ileum and Colon
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RNA was isolated and cDNA synthesized a described previously [43 (link)]. Briefly, the RNAlater stored cross-sections of the terminal ileum, and transversal colon was homogenized with 2 mm zirconia beads (BioSpec Products, Bartlesville, OK, USA) in TissueLyser LT beadbeater (Qiagen, Hilden, Germany) and total RNA isolated by the RNeasy Plus Mini kit (Qiagen). Of total RNA, 500 ng was reverse transcribed by QuantiTect Reverse Transcription kit (Qiagen). The synthesized cDNA was 1/10 diluted with PCR quality water (Life Technologies, Carlsbad, CA, USA), and these PCR templates were stored at −25 °C until the Real-Time PCR.
4
Amniotic Membrane RNA Extraction
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A piece of approximately 1 mg of amniotic membrane stored in RNAlater was transferred to 350 μL of RLT Plus buffer 2 of RNAeasy Micro Plus kit (Qiagen, Hilden, Germany) in 2 mL Eppendorf tube with 2 mm diameter zirconia beads (BioSpec Products, Bartlesville, OK, USA) and homogenized in TissueLyser LT beadbeater (Qiagen) at 50 Hz for 5 min. Further steps of the total RNA purification followed the RNAeasy Micro Plus kit manufacturer’s instructions. The purity of the RNA was evaluated as a ratio of absorbances at 260 and 280 nm. Its quantity was measured by Quant-iT™ RiboGreen® RNA Assay Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instruction on the fluorescence microplate reader Infinite M200 (Tecan, Grödig, Austria). Ten ng of total RNA were reverse transcribed with the QuantiTect Reverse Transcription kit according to manufacturer’s instruction (Qiagen). Eighty μL of PCR grade water (Life Technologies) was added to 20 μL of the cDNA mixture to prepare a template for quantitative PCR.
5
Intestinal and Lymphoid Tissue Sampling
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The jejunum (40 cm segment of the proximal part of the jejunum) and ileum (40 cm segment of a distal part of the small intestine containing the distal jejunum and the ileum) were filled with 2 mL of PBC, gently kneaded, and rinsed. The entire colon was cut into small pieces on a 90 mm Petri dish and lavaged in 4 mL of PBC. Pieces of mesenteric lymph nodes, liver, and spleen—1 part (g) of the tissue and four parts (mL) deionized water were homogenized in a 2 mL Eppendorf tube with 3.2 mm stainless steel beads in a TissueLyser LT BeadBeater (Qiagen, Hilden, Germany). The lavages, tissue homogenates, and blood were log 10 serially diluted in PBC. The cultivation of BB12 was performed in 50 mm Petri dishes with Wilkins–Chalgren agar (Oxoid) supplemented with soya peptone (5 g/L, Oxoid), L-cysteine (0.5 g/L, Merck), mupirocin (100 mg/L, Merck), and glacial acetic acid (1 mL/L, Merck). The Petri dishes were cultivated in anaerobic jars with AnaeroGen sachets (Oxoid) at 37 °C for 48 h. S. Typhimurium was cultivated aerobically in a 90 mm Petri dish with MacConkey agar (Merck) at 37 °C for 24 h. The BB12 and LT2 CFU were counted from the plates optimally containing 10–100 colonies and 20–200 colonies, respectively.
6
RNA Extraction and cDNA Synthesis from Tissue
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10 mg pieces of intestinal cross sections or mesenteric lymph nodes were stored in RNAlater (Sigma-Aldrich, St. Luis, MO). They were transferred to 2 ml Eppendorf tubes containing 350 µl of RLT Plus buffer of the RNAeasy Micro Plus kit (Qiagen, Hilden, Germany), 2 µl of antifoaming reagent DX (Qiagen), and 2 mm zirconia beads (BioSpec Products, Bartlesville, OK, USA). The samples were homogenized in the TissueLyser LT beadbeater (Qiagen) at 50 Hz for 8 min. The remainder of the total RNA purification followed the RNAeasy Micro Plus kit manufacturer’s instructions. The purity of the RNA was measured in 10 mM Tris-HCl buffer, pH 7.5, as a ratio of absorbances at 260 and 280 nm corrected at 320 nm. 500 ng of total RNA (with A260–A320/A280–A320 ≥ 2.00) were used for reverse transcription with the QuantiTect Reverse Transcription kit (Qiagen). The RNA template was preincubated at 42°C for 2 min with a genomic DNA wipeout buffer. The synthesis of cDNA was carried out using a mixture of random n-mers and oligo (dT) primers incubated at 42°C for 20 min. The reaction was stopped by heating the mixture at 95°C for 3 min. 180 µl of PCR-grade water (Life Technologies, Carlsbad, CA, USA) was added to 20 µl of the cDNA mixture to prepare cDNA template for quantitative PCR.
7
Intestinal Tissue RNA Extraction Protocol
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Cross-sections (1–2 mm) of terminal ileum and transversal colon were stored in RNAlater (Qiagen, Hilden, Germany) at −20 °C until RNA purification. Slices of the intestine were moved from RNAlater to 600 μL RLT buffer of the RNeasy Plus Mini kit (Qiagen) containing an antifoaming reagent DX (Qiagen) and 2 mm zirconia beads (BioSpec Products, Bartlesville, OK) in 2 mL Eppendorf tube. The tissue was homogenized in TissueLyser LT beadbeater (Qiagen) at 50 Hz for 5 min at RT. The next steps of the total RNA purification followed the manufacturer’s instructions. Total RNA (500 ng) with ratio absorbances A260−A320/A280−A320 ≥ 2.0 measured in 10 mM Tris-HCl buffer pH 7.5 were reverse transcribed by QuantiTect Reverse Transcription kit (Qiagen) with initial 2 min genomic DNA wipeout at 42 °C, 20 min reverse transcription at 42 °C, and 3 min terminating step at 95 °C according to manufacturer’s instructions. Then, 180 μL of PCR quality water (Life Technologies, Carlsbad, CA) was added to 20 μL of the synthesized cDNA, and these 1/10 diluted PCR templates were stored at −25 °C until the following real-time PCR.
8
RNA Extraction from Intestinal Tissue
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The terminal ileum and transverse colon cross-section slices and small pieces of mesenteric lymph nodes were put into RNAlater and stored at −20 °C. Later they were moved into the RTL buffer of RNeasy Mini Kit Plus (Qiagen) and homogenized with 2 mm zirconia beads (BioSpec Products, Bartlesville, OK, USA) in TissueLyser LT beadbeater (Qiagen). The total RNA was isolated according to the manufacturer’s protocol. A total of 500 ng of total RNA was reverse transcribed by QuantiTect Reverse Transcription kit (Qiagen). The prepared cDNA was 1/10 diluted by PCR quality water (Life Technologies, Carlsbad, CA, USA), and this cDNA template was stored at −25 °C till quantitative PCR was performed.
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