Kidney tissue biopsies were immersed in RNAlater solution (Ambion AM7020) and stored at -80 °C. Specimens were thawed slowly on ice, put into RNAlater, and microdissection was performed manually under a microscope. For every tissue sample, ∼80-100 glomeruli were released from their surrounding capsule and the remaining tissue was considered as tubule and was put in RNeasy RNA tissue lysis buffer solution (Qiagen #74106) as per the manufacturer’s instructions. The total RNA of 10 mg samples was isolated using Qiagen RNeasy kit (#74106) according to manufacturer’s instructions. Agilent Bioanalyzer RNA 6000 Pico kit (Agilent Technologies #5067-1513) was used to check RNA quality. All samples with an RNA integrity number (RIN) >6 were used for cDNA preparation. Strand specific RNA-seq libraries were generated using TruSeq RNA library prep kit v2 (#RS-122-2001) following the manufacturer’s protocol. RNA-seq libraries were sequenced to a depth of 20 million 2 × 150 pair end reads.
Rneasy rna tissue lysis buffer solution
Manufactured by Qiagen
The RNeasy RNA Tissue Lysis Buffer Solution is a reagent designed to facilitate the extraction and purification of RNA from various tissue samples. It is a component of the RNeasy RNA extraction kit and serves to effectively lyse and homogenize the tissue, releasing the RNA for subsequent purification steps.
Automatically generated - may contain errors
Lab products found in correlation
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Am7020, by Thermo Fisher Scientific (2 mentions)
Magattract high molecular weight dna kit, by Qiagen (2 mentions)
Agilent bioanalyzer rna 6000 pico kit, by Agilent Technologies (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
3 protocols using rneasy rna tissue lysis buffer solution
1
Transcriptomic Analysis of Kidney Glomeruli
2
Kidney Tissue Collection and Processing
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Human kidney tissue collection was approved by the University of
Pennsylvania Institutional Review Board. Kidney samples were obtained from
surgical nephrectomies. Nephrectomies were de-identified, and the corresponding
clinical information was collected through an honest broker; therefore, no
consent was obtained from the subjects.
Collected tissue was immersed in RNAlater (Ambion#AM7020) solution at
4°C for several hours prior to being stored at −80°C in
RNAlater. Tissue was thawed on ice, placed in RNAlater, and manually
microdissected for glomerular and tubular compartments. In general,
60–150 glomeruli that readily released from the capsule were collected
and placed into RNeasy RNA Tissue Lysis Buffer Solution (per Qiagen RNeasy kit
manufacturer instructions (Qiagen#74106)). We refer to the remaining compartment
as tubule throughout the article.
Part of the tissue core was formalin fixed and paraffin embedded. These
samples were later sectioned and stained with Hematoxylin eosin or periodic acid
schiff. Our local renal pathologist performed an unbiased review of the tissue
section by scoring multiple parameters.
DNA was isolated using the Qiagen DNAeasy or MagAttract High Molecular
Weight DNA Kit (Qiagen#67563) according to the manufacturer’s
instructions. DNA was quantified by the Invitrogen Quant-iT PicoGreen dsDNA
Assay Kit (Invitrogen#P11496).
Pennsylvania Institutional Review Board. Kidney samples were obtained from
surgical nephrectomies. Nephrectomies were de-identified, and the corresponding
clinical information was collected through an honest broker; therefore, no
consent was obtained from the subjects.
Collected tissue was immersed in RNAlater (Ambion#AM7020) solution at
4°C for several hours prior to being stored at −80°C in
RNAlater. Tissue was thawed on ice, placed in RNAlater, and manually
microdissected for glomerular and tubular compartments. In general,
60–150 glomeruli that readily released from the capsule were collected
and placed into RNeasy RNA Tissue Lysis Buffer Solution (per Qiagen RNeasy kit
manufacturer instructions (Qiagen#74106)). We refer to the remaining compartment
as tubule throughout the article.
Part of the tissue core was formalin fixed and paraffin embedded. These
samples were later sectioned and stained with Hematoxylin eosin or periodic acid
schiff. Our local renal pathologist performed an unbiased review of the tissue
section by scoring multiple parameters.
DNA was isolated using the Qiagen DNAeasy or MagAttract High Molecular
Weight DNA Kit (Qiagen#67563) according to the manufacturer’s
instructions. DNA was quantified by the Invitrogen Quant-iT PicoGreen dsDNA
Assay Kit (Invitrogen#P11496).
3
Kidney Tissue Collection and Processing
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