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:
- Add 1/10 volume 3 M NaOAc, pH 5.2, and 2.5 volumes ethanol.*
- Mix and incubate at -20ºC for at least 1 hour.
- Centrifuge at .12,000 x g in a microcentrifuge for 20 minutes at 4ºC.
- Wash pellet twice with 80% ethanol.
- Air dry pellet. Check for dryness before proceeding.
- 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.
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.