Frozen spleen samples (30 mg) were transferred into RNAlater™-ICE Frozen Tissue Transition Solution (Thermo Fisher Scientific, Waltham MA, USA) and stored overnight at 4°C to prevent mRNA degradation while thawing. Next day, spleen tissue was ground in liquid nitrogen using a mortar and pestle, then homogenized using shredder columns (Qiagen, Germantown, MD, USA). RNA was purified using RNeasy Protect kit (Qiagen, Germantown, MD, USA). Purity and concentration of RNA was analyzed in a nanophotometer (IMPLEN P330) at absorbances A260/280 (protein contamination) and A260/230 (buffer contamination) before proceeding to cDNA preparation.
Shredder columns
Manufactured by Qiagen
Sourced in United States
Shredder columns are a laboratory equipment used for homogenizing and disrupting solid or semi-solid samples. The columns mechanically break down the sample material to facilitate extraction, purification, or analysis of target molecules or components.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rneasy protect kit, by Qiagen (1 mentions)
Rnalater ice frozen tissue transition solution, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
3 protocols using shredder columns
1
Spleen RNA Extraction Protocol
2
RNA Extraction from Frozen Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
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After collection, all tissues except bone marrow were snap frozen in liquid nitrogen and stored at –80 °C until used. Frozen tissues were mechanically homogenized with 1 stainless steel bead (5 mm) in 1 ml Trizol (Thermo Fisher Scientific) by shaking for 50 s at 30 Hz (TissueLyser, Qiagen). 200 μL chloroform was added to homogenize Trizol samples, followed by 15 s shaking and centrifugation (15 min, 12000 RCF, 4 °C). The chloroform phase was collected, shaken for 15 s with 500 μL isopropanol and centrifuged (50 min, 12,000 RCF, 4 °C). The pellet was washed with 70% ethanol twice (10 min, 8000 RCF, 4 °C) and dissolved in 50 μL PCR-grade water. Bone marrows were flushed immediately after collection from mouse, cells were spun and loaded onto shredder columns (Qiagen). Shredded bone marrow cells were frozen (–80 °C) in RLT buffer (1% beta-mercaptoethanol) until RNA extraction using RNAeasy mini kit (Qiagen). RNA integrity number (RIN) was determined in all samples (Bioanalyzer 2100, Agilent) before sequencing.
3
Liver RNA Isolation and Purification
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