About Quick-RNA™ MidiPrep
The Quick-RNA™ MidiPrep is an innovative product designed for the easy, reliable, and rapid isolation of total RNA from cultured cells or solid tissue samples. The procedure combines a unique, single-step RNA extraction/binding buffer with Fast-Spin column technology to yield high quality RNA in minutes. The method is easy: simply add the provided ZR RNA Buffer to extract total RNA from the cells of interest and then purify the RNA using the provided spin column. The result is highly-concentrated, purified RNA that is suitable for subsequent RNA-based methods including RT-PCR, hybridization, etc.
|Processing Time||15 min|
|Sample Sources||Cells from culture or tissue samples, buffy coat, plasma, serum, and other biological liquids. Not compatible with whole blood. (For purification of RNA from whole blood see the ZR Whole-Blood RNA MiniPrep (R1020))|
|RNA Purity||High quality RNA (A260/A280 > 1.8, A260/A230 > 1.8) suitable for all downstream RNA-based manipulations.|
|RNA Recovery||Up to 1 mg RNA can be eluted into ≥ 200 µl RNase-free water allowing for a highly concentrated sample.|
|Sample Size||~103 to 108 cells.|
|Equipment Needed||Vacuum manifold, microcentrifuge.|
Human RNA isolated from cell culture with the Quick-RNA™ MiniPrep Kit. M: 1 kb DNA marker (Zymo Research); 1, 2: duplicate RNA preparations.
PCR amplification of a ß-actin transcript (353 bp fragment shown) following RNA isolation from human epithelial cells (HCT 116) with the Quick-RNA™ MidiPrep: RT-PCR (lanes 1, 2), PCR negative control (RNA template; lanes 3, 4). M1 and M2 are 100 bp and 1 kb DNA Markers, respectively (Zymo Research).
Sample Homogenization and Cell Lysis: Follow an applicable method (A-D).
A. Adherent Cells: Cells can be lysed directly in the culture container by removing liquid medium and adding ZR RNA Buffer1 directly to the monolayer (e.g., 3 ml for 5x106 cells). Remove cells from culture surface by pipetting, scraping, etc.
B. Cells in Suspension: Pellet cells by gentle centrifugation (e.g., 5 minutes at 500 x g). Remove the supernatant completely and resuspend the cell pellet in 3 ml ZR RNA Buffer1. Vortex briefly.
C. Solid Tissue Samples: Add 3 ml ZR RNA Buffer1 to fresh or frozen tissue (up to ~100 mg) and homogenize the sample (e.g., using a Dounce or similar homogenizer). Important! To eliminate tissue debris, if present, sample lysates may be spun at ≤500 x g for 1 minute.
D. Liquid Samples: Add 3 volumes of ZR RNA Buffer1 to every volume of sample (e.g., 3 ml of buffer to 1 ml sample).
Transfer lysate into the Zymo-Spin™ V-E Column with Reservoir mounted on a vacuum manifold and start vacuum4.
Remove the reservoir and transfer the Zymo-Spin™ V-E Column into a Collection Tube. Centrifuge the column at ≥12,000 x g for 30 seconds.
Add 400 µl RNA Pre-Wash Buffer to the column and centrifuge at ≥12,000 x g for 1 minute. Discard the flow-through.
Add 400 µl RNA Wash Buffer to the column and centrifuge at ≥12,000 x g for 30 seconds. Discard the flow-through and repeat Step 5.
Centrifuge the Zymo-Spin™ V-E Column at ≥12,000 x g for 2 minutes in the emptied Collection Tube to ensure complete removal of the wash buffer.
Place the Zymo-Spin™ V-E Column into an RNase-free tube. Add ≥200 µl DNase/RNase-Free Water5 directly to the column matrix and let stand at room temperature for 1 minute. Centrifuge at top speed for 1 minute. Eluted RNA can be used immediately or stored at ≤-70 ºC.