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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
Recommended RNA Preparation
Methods:
Cultured Cells and Fine Needle Aspiration Biopsies (FNAB):
Use our ArrayGrade™ Total RNA Isolation Kit (GA-013).
Tissues:
First, extract RNA from the tissue using the TRIzol® protocol (Invitrogen,
Catalog # 15596-026). Be sure to use a sufficient amount of TRIzol® reagent.
During homogenization, add a volume of reagent at least ten times greater than
the tissue volume.
Then after the ethanol precipitation step, further clean up the RNA using our ArrayGrade™
Total RNA Isolation Kit (GA-013).
Whole Blood
Samples:
Before RNA preparation, remove red blood cells (RBC) from whole blood samples
using a density gradient centrifugation medium (for example, Lymphoprep, Greiner
Bio-One, Catalog # 1031966). Use our ArrayGrade™
Total RNA Isolation Kit (GA-013)
to isolate total RNA from the white blood cell fraction. Alternatively, the
PAXgene Blood RNA Kit (Qiagen, Catalog # 762134) can also be used to prepare
total RNA from whole blood samples.
NOTE: Total RNA
Isolated Using a Phenol-Based Method:
Total RNA already prepared from any biological source material using a
phenol-based method (such as TRIzol, RNAzol, etc.) must be further purified with
our ArrayGrade™ Total RNA Isolation Kit (GA-013).
Formalin-Fixed
Paraffin-Embedded (FFPE):
Use our ArrayGrade™ FFPE RNA Isolation Kit
(GA-023).
Laser Capture
Microdissection (LCM):
Please consult a Technical Support representative before isolating and
submitting samples. Capture cells on Arcturus Capsure® LCM caps (catalog number
LCM0211), and then use the Arcturus PicoPure® RNA Isolation Kit (Catalog Number
KIT0204) to isolate the RNA.
Other
Biological Samples:
Refer to existing literature to find isolation protocols for high-quality RNA
from other biological samples or contact a Technical Support representative.
Suspend total RNA
samples in RNase-free water (not DEPC-treated water) or RNase-free 10 mM Tris
buffer pH 8.0.
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).
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