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Detecting Attomole Amounts of Small RNA mirVana™ miRNA
Detection Kit
•Sensitive -- Detect
miRNA or siRNA in as little as 10 ng total RNA
•Specific -- Extremely
low background with no need to optimize hybridization or washing
conditions
•Simple and fast
-- Single tube procedure eliminates the need to transfer to
membrane for hybridization
•Multiple target
detection-- Detect small RNAs and mRNAs in the same sample
The new mirVana miRNA Detection Kit
provides a faster and more sensitive alternative to Northern
analysis for detecting small RNAs. With this patented technology,
quantitation of small RNA species can be performed with as little
as 10-50 ng of total RNA. The assay is incredibly sensitive and
is able to detect attomole (10-18 mol) amounts of
target RNA. In addition, the mirVana miRNA Detection Kit
can be used to simultaneously detect several small RNAs of the
same size or both small RNA and long RNA species in the same
sample. This means you can now quantitate an siRNA and its target
mRNA in the same sample. (See several applications of this kit
in Detecting miRNA & siRNA.)
Rapid Procedure Based on Solution Hybridization
The mirVana miRNA Detection Kit
is based on a simple solution hybridization principle. The sample
RNA containing the target RNA(s) of interest is mixed
with one or more high specific activity radiolabeled RNA probes and the included
Hybridization Buffer. After heat denaturation, each mixture is incubated at
42°C to hybridize the probe to its complementary RNA molecules. Unhybridized
RNA species and excess RNA probe is then removed by a rapid ribonuclease digestion
step. The hybridized, protected RNA fragments are recovered in the same tube
using Ambion's patented single step technology for simultaneous ribonuclease
inactivation and nucleic acid precipitation. RNA samples are then resuspended
with the provided Gel Loading Buffer and analyzed on a denaturing polyacrylamide
gel. Because the hybridization is performed in solution, the procedure ensures
a sensitive and linear detection signal after autoradiography. Furthermore,
the included Hybridization Buffer has been specifically developed to provide
the
optimal sensitivity and specificity of detection with short antisense probes.
Thus the procedure is ideal for detecting small RNA molecules such as siRNA
or miRNA.
Complete Kit with Included Controls
Each mirVana miRNA Detection
Kit provides enough reagents for 100 reactions. The kit also
contains a control transcription template and Probe Elution
Buffer
to prepare and gel purify a 32 nt RNA probe specific for miR-16 miRNA. When
used with the provided Mouse Kidney Total RNA, this probe generates a 22 nt
protected fragment. The kit also comes with a detailed Instruction Manual and
comprehensive information about experimental setup and probe design/preparation.
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| Figure 1. Differential
Recovery of Small RNAs During Total RNA Isolation. Total
RNA was isolated from 1x106 HeLa cells using
three standard RNA isolation procedures: single phenol/guanidinium
extraction (RNAwiz™), glass fiber filter (RNAqueous®) purification,
or double phenol extraction (ToTALLY RNA™).
Purified RNA samples were analyzed on a 1% denaturing
agarose gel and recovery of messenger RNA (ß-actin,
GAPDH), small nuclear RNA (U1 snRNA), and miRNA (miR-16,
miR-22) were assessed by Northern blot or solution hybridization
with the mirVana miRNA Detection
Kit. |
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