|
Now Available: TRI Reagent®
RNA, DNA, and Protein
from a Single Sample
• Use one reagent to isolate
DNA-free RNA, RNA-free DNA, and/or protein
• Obtain higher yields than
traditional guanidine thiocyanate or cesium chloride methods
• Compatible with many sample
sources (e.g., human, plant, yeast, bacterial, or viral samples)
• Easily scalable depending
on starting material and amountof nucleic acid or protein needed
• Fast, simple complete sample
processing in ~1 hour
TRI Reagent® (patented product/reagent)
is a ready-to-use reagent for the isolation of total RNA or
the simultaneous isolation of RNA, DNA, and protein from diverse
biological samples. During a one-step sample homogenization/
lysis procedure, TRI Reagent disrupts cells and denatures endogenous
nucleases. The homogenate is separated into aqueous and organic
phases by addition of bromochloropropane (or chloroform) followed
by centrifugation (see sidebar, Advantages
of Using Bromochloropropane Instead of Chloroform). RNA
partitions to the aqueous phase, DNA to the interphase, and protein
to the organic phase.
This highly reliable technique performs well
with both small and large quantities of tissues and cultured
cells (e.g., samples larger than ~5 mg tissue or 5 x 105 cultured
cells) (Figure 1). RNA isolated using TRI Reagent is suitable
for use in real-time and end-point RT-PCR, amplification/labeling
for microarray analyses, in vitro translation, cDNA synthesis,
Northern blotting, and RNase protection assays. RNA preparations
isolated using bromochloropropane often do not require an additional
DNase treatment, but Ambion's
TURBO DNA-free Kit can be used to remove residual genomic DNA before
RT-PCR assays. In addition, PCR, Southern blotting, and cloning can be performed
using DNA, and denaturing polyacrylamide gel electrophoresis, and Western blot
assays can be performed using protein isolated with TRI Reagent. See www.ambion.com/prod/tri for more information and detailed protocols.
|
|
|
Figure
1. Bromochloropropane (Phase Separation
Reagent) Improves Genomic DNA Removal in the TRI Reagent® Single-Step
RNA Isolation Method. Total RNA was isolated
from RNAlater®-treated mouse liver, kidney,
brain, lung, heart, and spleen and from fresh cultured
human cells using the standard TRI reagent protocol with
either bromochloropropane (BCP) or chloroform (CHCl3).(A) Genomic
DNA removal was assessed by one-step, (+) or (–)
RT, quantitative PCR targeting mouse or human TATA binding
protein (TBP) mRNA and gene sequences, and is presented
as percent of residual gDNA. (B) Total
RNA yield was determined by A260 using the NanoDrop® spectrophotometer,
and RNA yield (µg/mg) was normalized to mg of mouse
tissue input or to 0.5 x 105 human cultured cells.
|
Scientific Contributors
Quoc Hoang, WeiWei Xu, Roy Chris Willis, Angela Burrell, Mangkey
Bounpheng, Xingwang Fang • Ambion, Inc.
back
to top
|
