RNA Isolation : NIDDK

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RNA Isolation


Sample preparation is integral to performing a good microarray (or any type of quantitative) experiment. For conventional methods and requirements for Affymetrix experiments, see RNA isolation protocol (below). For more robust RNA handling, here are some suggestions:

Ideally, one should stabilize RNA from the moment of harvesting samples. This kit from Qiagen addresses the issues with regards to immediate RNA stabilization and isolation in a single kit: RNeasy Protect Mini, Midi, and Maxi Kits

Tissue Storage

When an RNA extraction protocol has not been fully optimized and you´re ready to collect samples: An alternative to freezing tissue samples in liquid nitrogen or preparing RNA from source tissue/cells immediately, is to stabilize and protect cellular RNA in samples with RNAlater. This procedure is also ideal for experiments where many samples are collected over several time points. Tissue pieces or cells can be harvested and submerged in RNAlater for storage (frozen or unfrozen) while retaining the original quality and quantity of RNA with subsequent RNA isolation.

For animal cells, use RNAlater TissueProtect Tubes and RNAlater RNA Stabilization Reagent

For bacterial cells, use RNAprotect Bacteria Reagent

Small sample RNA

For those interested in isolating RNA from small samples:

Stratagene has two products that are useful for small scale RNA isolation typically when LCM samples are used as starting material: Absolutely RNA Microprep kit (up to 5 x 10^5 cells) and the Absolutely RNA Nanoprep kit (single cell to 5 x 10^4 cells). Each procedure allows the end user to use a small elution volume 10-30 ul so the RNA can be used in downstream applications such as RT-PCR, QRT-PCR and microarray target labeling without having to concentrate it via EtOH precipitation.

Absolutely RNA Microprep kit manual (PDF)
Absolutely RNA Nanoprep kit manual (PDF)

Qiagen also carries a kit for isolation of concentrated total RNA from small amounts of tissue (<5 mg) or small numbers of cells (<5 x 10^5 up to a single cell): RNeasy Micro Kit

For special tissue types

For isolation of total RNA from fiber-rich tissues such as heart, muscle, esophagus, skin, use: RNeasy Fibrous Tissue Mini and Midi Kits

For isolation of total RNA from fatty tissues such as brain, adipose tissue, use: RNeasy Lipid Tissue Mini and Midi Kits

Link to all of Qiagen´s RNA isolation protocols: Literature

Recommended Protocols for RNA Isolation

Materials for Total RNA Isolation

_ TRIzol Reagent, Invitrogen Life Technologies, P/N 15596-018
_ RNeasy Mini Kit, QIAGEN, P/N 74104

Isolation of RNA

The quality of the RNA is essential to the overall success of the analysis. Since the most appropriate protocol for the isolation of RNA can be source dependent, it is recommended to use a protocol that has been established for the tissues or cells being used. In the absence of an established protocol, use one of the commercially available kits designed for RNA isolation. When using a commercial kit, follow the manufacturer´s instructions for RNA isolation.

Isolation of Total RNA from Yeast

Good quality total RNA can be isolated from yeast cells using a hot phenol protocol described by Schmitt, et al. Nucl Acids Res, 18:3091-3092 (1990).

Isolation of Total RNA from Arabidopsis

TRIzol Reagent from Invitrogen Life Technologies is used to to isolate total RNA from Arabidopsis. Please follow the instructions provided by the supplier and, when necessary, use the steps outlined specifically for samples with high starch and/or high lipid content.

Isolation of Total RNA from Mammalian Cells or Tissues

High-quality total RNA can be isolated from mammalian cells (such as cultured cells and lymphocytes) using the RNeasy Mini Kit from QIAGEN. If mammalian tissue is used as the source of RNA, total RNA can be isolated with a commercial reagent such as TRIzol.

Some points to consider before starting:
RNeasy protocol starts with a cell pellet concentration of 1x107 cells/ml or less.
RNA should be isolated from cells ASAP after harvesting.
If cells or tissue need to be stored after harvesting they should be suspended in RNAlater (Ambion cat.# 7020) to prevent degradation of RNA by RNAse activity.
See RNeasy Kit Manual for tissue disruption methods.

If going directly from TRIzol-isolated total RNA to cDNA synthesis, it may be beneficial to perform a second cleanup on the total RNA before starting. After the ethanol precipitation step in the TRIzol extraction procedure, perform a cleanup using QIAGEN RNeasy Mini Kit. Much better yields of labeled cRNA are obtained from the in vitro transcription-labeling reaction when this second cleanup is performed.

Quantification of RNA

Quantify RNA yield by spectrophotometric analysis using the convention that 1 absorbance unit at 260 nm equals 40 µg RNA per mL. The absorbance should be checked at 260 and 280 nm for determination of sample concentration and purity. The A260 /A280 ratio should be close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable). ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Affymetrix recommends starting the cDNA synthesis protocol with a minimum of 0.2 µg poly-A mRNA at a minimum concentration of 0.02 µg/µL, or 5 µg of total RNA* at a minimum concentration of 0.5 µg/µL, in order to obtain sufficient quantity of labeled cRNA for target assessment and hybridization to GeneChip expression probe arrays. There are two major advantages to starting with at least the recommended amount of material:

1. Enough material to check sample yield and quality at the various steps of this protocol.
2. Production of enough cRNA for hybridization of the target to multiple probe arrays.

* We prefer to start with at least 5 µg to ensure sufficient yield of good quality cRNA for hybridization


Precipitation of Total RNA

It is not necessary to precipitate total RNA following isolation or cleanup with RNeasy Mini Kit.

Please adjust elution volumes from the RNeasy column to prepare for cDNA synthesis based upon expected RNA yields from your experiment. Ethanol precipitation is required following TRIzol isolation and hot phenol extraction methods:

Precipitation Procedure

  1. Add 1/10 volume 3 M NaOAc, pH 5.2, and 2.5 volumes ethanol.*
  2. Mix and incubate at -20ºC for at least 1 hour.
  3. Centrifuge at .12,000 x g in a microcentrifuge for 20 minutes at 4ºC.
  4. Wash pellet twice with 80% ethanol.
  5. Air dry pellet. Check for dryness before proceeding.
  6. Resuspend pellet in DEPC-treated H2O. The appropriate volume for resuspension depends on the expected yield and the amount of RNA required for the cDNA synthesis. Please read ahead to the cDNA synthesis protocol in order to determine the appropriate resuspension volume at this step.

*Addition of Carrier to Ethanol Precipitations

Adding carrier material has been shown to improve the RNA yield of precipitation reactions.

Pellet Paint

Affymetrix has found that adding 0.5 µL of Pellet Paint per tube to nucleic acid precipitations makes the nucleic acid pellet easier to visualize and helps reduce the chance of losing the pellet during washing steps. The pellet paint does not appear to affect the outcome of subsequent steps in this protocol; however, it can contribute to the absorbance at 260 nm when quantifying the mRNA.


Addition of 0.5 to 1 µL of glycogen (5 mg/mL) to nucleic acid precipitations aids in visualization of the pellet and may increase recovery. The glycogen does not appear to affect the outcome of subsequent steps in this protocol.

Page last updated: January 01, 0001

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