About ZR RNA MiniPrep™
The ZR RNA MiniPrep™ provides a quick method for high quality total RNA isolation from cells, needle biopsies, and tissue. The product isolates both large and small RNA species without the use of reducing agents or phenol. Small RNAs (e.g., tRNAs, microRNAs) can be recovered with a simple adjustment of the RNA isolation protocol. Eluted RNA is suitable for use in RT-PCR and other RNA-based procedures.
| Format | Spin Column |
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| Processing Time | 15 min |
| Equipment | Microcentrifuge |
| Sample Sources | Cells from culture or small amounts of solid tissue. |
| Storage | ≤-70ºC. The addition of RNase inhibitors is optional but highly recommended for prolonged storage. |
| RNA Purity | High quality RNA (A260/A280>1.8, A260/A230>1.8) is recovered. |
| RNA Recovery | RNA can be eluted into small volumes, ≥ 25 µl allowing for a highly concentrated sample. Maximum RNA binding capacity of provided column is ~25 µg. |
| Sample Size | 102 - 107 cells in suspension or solid form. |
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Recovery of small RNA. Total RNA isolated using the ZR RNA MicroPrep™ was resolved in an agarose gel (2-4) and small RNAs from the same sample were also resolved in a native polyacrylamide gel (6-7). Input = 105 yeast cells spiked with 1 µg ZR small-RNA™ Ladder (Zymo Research)
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PCR amplification of ß-actin transcript post-RT (353 bp fragment shown): Total RNA from human epithelial cells (HCT 116) was isolated using the ZR RNA MicroPrep™.
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| Step 1 | Cell homogenization/sample preparation2: a. Cell samples: Isolate cells by gentle centrifugation and remove the supernatant. Resuspend the pelleted cells in 400 µl RNA Lysis Buffer b. Tissue samples: Add 400 µl RNA Lysis Buffer directly to the sample and mechanically homogenize up to 25 mg fresh or frozen tissue. c. Liquid samples/suspensions: Add 4 volumes RNA Lysis Buffer to the sample and mix well (e.g., 320 µl buffer added to 80 µl sample). Adjust the reagents volume proportionally as needed. |
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| Step 2 | Centrifuge the sample mixture at ≥12,000 × g for 1 minute |
| Step 3 | Transfer the lysate (i.e., the supernatant from Step 2) to a Zymo-Spin™ IIIC Column in a Collection Tube. Centrifuge at 8,000 × g for 30 seconds. Save the flow-through! |
| Step 4 | Add 0.8 volume ethanol (95‑100%) to the flow-through in the Collection Tube and mix well (e.g., 320 µl ethanol added to 400 µl flow-through). For quantitative small RNA recovery, use 2 volumes ethanol (95‑100%)3. |
| Step 5 | Transfer the mixture to a Zymo-Spin™ IIC Column4 in a Collection Tube. Centrifuge at ≥12,000 × g for 1 minute5. Discard the flow-through. |
| Step 6 | Add 400 µl RNA Prep Buffer to the column. Centrifuge at ≥12,000 × g for 1 minute. Discard the flow-through and replace the Zymo-Spin™ IIC Column back into the Collection Tube. |
| Step 7 | Add 800 µl RNA Wash Buffer to the column. Centrifuge at ≥12,000 × g for 30 seconds. Discard the flow-through and place the Zymo-Spin™ IIC Column back into the Collection Tube. Repeat the wash step with 400 µl RNA Wash Buffer. |
| Step 8 | Centrifuge Zymo-Spin™ IIC Column at ≥12,000 × g for 2 minutes in the emptied Collection Tube to ensure complete removal of the wash buffer. |
| Step 9 | Place the Zymo-Spin™ IIC Column into an RNase-free tube. Add ≥25 µl DNase/RNase-Free Water directly to the column matrix and let stand at room temperature for 1 minute. |
| Step 10 | Centrifuge at 10,000 × g for 30 seconds to elute the RNA from the column. RNA can be used immediately or stored at ≤-70 oC. |
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“Speed!!! This was a wonderful time saver over my usual TriZol/BCP method. While I've never had much problem with gDNA contamination, my samples were both high concentration and free of gDNA following the miniprep.”
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Aaron B. (University of Tennessee, Knoxville)
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