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Recommended RNA Isolation Methods

RNA samples are very sensitive to RNase digestion.
Wear gloves and maintain an RNase-free work area while isolating RNA.

Recommended RNA Preparation Methods:

Cultured Cells and Fine Needle Aspiration Biopsies (FNAB)
Tissues
Whole Blood
Formalin-Fixed Paraffin-Embedded (FFPE)
Laser Capture Microdissection (LCM)
Other Biological Samples

Recommended RNA Quality Control

1. Recommended RNA Preparation Methods:
High quality total RNA for your real-time PCR experiment must be prepared using one of the following methods, each specific for your biological sample:
a. Cultured Cells:
Use the Qiagen RNeasy® Mini Kit (Catalog # 74104).
" You must perform the recommended on-column DNase treatment step (RNase-
Free DNase Set: 79254).
b. Tissue Samples:
i. First, extract RNA from the tissue using the QIAzol/TRIzol® protocol. Be sure to
use a sufficient amount of QIAzol/TRIzol reagent. During homogenization, add a
volume of reagent at least ten times greater than the tissue volume.
ii. After the ethanol precipitation step, further clean up the RNA using the Qiagen
RNeasy Mini Kit (Catalog # 74104).
" You must perform the recommended on-column DNase treatment step (RNase-
Free DNase Set: 79254).
c. Formalin-fixed Paraffin-embedded (FFPE) Samples:
i. Please refer to protocol for SABiosciences RT2 FFPE RNA Extraction Kit
(Catalog # PA-023/330471)
d. Small Samples Yielding Less than 100 ng Total RNA:
i. Please refer to protocol for Molecular Devices® PicoPure® RNA Isolation Kit
(Catalog # KIT0204)
e. Whole Blood Samples:
i. Before RNA preparation, red blood cells (RBC) must be removed from whole
blood samples using a density gradient centrifugation medium (for example,
Lymphoprep®, Greiner Bio-One, Catalog # 1031966).
ii. The white blood cell fraction is then used for RNA isolation with the Qiagen
RNeasy® Mini Kit (Catalog # 74104).
" You must perform the recommended on-column DNase treatment step.
iii. Alternatively, the PAXgene™ Blood RNA Kit (Qiagen, Catalog # 762164) can
also be used to prepare total RNA from whole blood samples.
f. Total RNA Isolated Using a Phenol-Based Method:
If you have already prepared total RNA from any biological source material using
a phenol-based method (such as QIAzol,TRIzol®, RNAzol, etc.), you must clean
up the RNA with the Qiagen RNeasy® Mini Kit (Catalog # 74104).
" You must perform the recommended on-column DNase treatment step (RNase-
Free DNase Set: 79254).
g. For Other Biological Samples:
Refer to the existing literature to find isolation protocols for high-quality RNA from
other biological samples or contact one of our Technical Support representatives.

For best results from the PCR Array, all RNA samples should be suspended in the RNase-free water provided with the RNA Isolation kit. DO NOT use DEPC-treated water!

Recommended Quality Control:

NOTE: Confirmation of the quantity and quality of LCM and other small sample sizes cannot be performed on the RNA itself. For more information, see our Complete Service Solution for LCM and other small sample sizes.

RNA Concentration and Purity by UV Spectrophotometry:
Measured in RNase-free 10 mM Tris, pH 8.0 buffer
Total RNA concentration by A260 should be greater than 0.5 mg/ml
A260:A280 ratio should be greater than 2.0.
A260:A230 ratio should be greater than 1.7.

Ribosomal RNA band integrity

Electrophorese a fraction of each RNA sample on a denaturing agarose gel or on an Agilent BioAnalyzer® using an RNA 6000 Nano LabChip® and verify that there is a sharp distinction at the small side of both the 18S and 28S ribosomal RNA (rRNA) bands or peaks. Any smearing or shoulder to the rRNA bands or peaks indicates that degradation has occurred in the RNA sample. For some sample results, see below.

NOTE: Due to the fragmentation associated with formalin fixation and paraffin embedding, the RNA isolated from FFPE blocks and slides is always degraded, and the extent or nature of that degradation varies among samples. Therefore, while the integrity of RNA isolated from FFPE blocks or slides may and should still be determined by the methods described here only for the purposes of comparing samples, the results of that analysis will not appear as described and will not otherwise be easily interpreted.

Figure: Good Ribosomal RNA Band Integrity Is Important for Best Results from the Gene Expression Analysis Services. Panel A displays an Agilent BioAnalyzer® electropherogram of a high-quality total RNA preparation showing sharp peaks without shoulders (especially to the left of each peak) for the 18S and 28S ribosomal RNA (left to right). Panel B, right-hand lane, displays an analysis of the same high-quality total RNA preparation by agarose gel electrophoresis demonstrating sharp bands (especially at the bottom of each band) for the 28S and 18S ribosomal RNA (top to bottom).