Rnasimple Total Rna Kit

RNAsimple Total
RNA Kit
For purification of high pure total RNA
www.tiangen.com/en
RP110413
RNAsimple Total RNA Kit

Cat. no. DP419
Kit Contents
DP419
Contents
50 preps
Buffer RZ 60 ml
Buffer RD 12 ml
Buffer RW 15 ml
RNase-Free ddH2O 15 ml
RNase-Free Spin Columns CR3 50
RNase-Free Collection Tubes 1.5 ml 50
RNase-Free Collection Tubes 2 ml 50
Handbook 1
Storage
Buffer RZ should be stored protected from light at 2-8°C; others
stored at room temperature (15-25°C).
Introduction
RNAsimple Total RNA Kit uses a new RNA isolation technology
based on Guanidine Thiocyanate / Phenyl method. It contains a
unique buffer RZ that minimizes the contamination of genomic
DNA and protein. RNAsimple Total RNA Kit can efficiently isolates
high pure RNA from blood, cells, tissues and plant samples in one
hour. The purified RNA is ready-to-use in downstream applications
such as: RT-PCR and real-time RT-PCR, gene-chips assay, northern
blot, dot blot, polyA screening, in vitro transcript, and molecular
cloning.
RNAsimple Total RNA Kit Handbook | 1

Important Note
For isolation of bacterial RNA, RNAprep Pure Kit (For Cell/Bacteria)
should be used (Cat.no. DP430).
Notes of preventing RNase contamination

1. Change gloves regularly. Bacteria on the skin can result in
RNase contamination.
2. Use RNase-Free plastic and tips to avoid cross contamination.
3. RNA can be protected in TRNzol. But RNA must be stored or
processed in RNase-Free plastic or glassware. To wipe off
RNase, the glassware can be roasted at 150℃ for 4 hours,
while plastic can be dipped in 0.5 M NaOH for 10min, washed
by RNase-Free ddH2O thoroughly, and sterilized.
4. Use RNase-Free ddH2O to confect solution. (Add DEPC into
water in clean glass container to a final concentration of 0.1%
(v/v). Incubate overnight and autoclave for 15 min to remove
any trace of DEPC.)
Protocol
Buffer RD and Buffer RW are supplied as a concentrate. Before
using for the first time, add ethanol (96–100%) as indicated on the
bottle to obtain a working solution.
1. Homogenizing samples.
a. Plant (take leaves as an example): Place fresh leaves in
liquid nitrogen and grind thoroughly with a mortar and
pestle, or grind in Buffer RZ after cut leaves into pieces.
This process is suggested to be finished within one minute.
Use 1 ml Buffer RZ per 100 mg leaves.
b. Tissues (take rat liver as an example): Add 1 ml Buffer RZ
for per 30–50 mg of liver sample. Homogenize sample

using a power homogenizer. Usually, the volume of tissue
sample should not exceed 10% of the volume of Buffer RZ.
7
c. Adherent Cells (do not use more than 1 × 10 cells): Cells
grown in a monolayer in cell-culture vessels can be either
lysed directly in the vessel (up to 10 cm diameter) or
trypsinized and collected as a cell pellet prior to lysis. (Cells
grown in a monolayer in cell-culture flasks should always
be trypsinized.)
1) Method A: To lyse cells directly. Add 1 ml Buffer RZ
2
directly to the cells in the culture dish per 10 cm of
culture dish surface area. Pipette the lysate up and
down several times. Note: the volume of Buffer RZ
should be determined according to the surface area
instead of the number of cells. An insufficient volume
can result in DNA contamination of isolated RNA.
2) Method B: To trypsinize and collect cells. Determine
the number of cells. Aspirate the medium, and wash
the cells with PBS. Aspirate the PBS, and add 0.10–
0.25% trypsin in PBS. After the cells detach from the
dish or flask, add medium (containing serum to
inactivate the trypsin), transfer the cells to an RNase-
Free glass or polypropylene centrifuge tube (not
supplied), and centrifuge at 300 x g for 5 min.
Completely aspirate the supernatant.
Note: Make sure that the supernatant has been
completely removed. Residual medium could lead to
incomplete lysis of cells and reduced yield of RNA.
d. Suspension Cells: Harvest cells by centrifugation and
6
remove culture medium. Add 1 ml of Buffer RZ per 5 × 10 -
7 7
10 cells from animal, plant or yeast, or 1 × 10 cells of
bacterial. Do not wash cells before addition of Buffer RZ to

avoid increased chance of mRNA degradation. Samples
from some yeast and bacteria maybe need to be
homogenized by using a power homogenizer.
e. Blood: Take fresh blood, and add three volumes of Buffer
RZ. Mix thoroughly. (Recommended amount: 0.75 ml
Buffer RZ for 0.25 whole blood)
2. Incubate homogenized samples at 15-30℃ for 5 min, to
permit complete dissociation of the nucleoprotein complex.
3. Optional step: Centrifuge the sample at 12,000 rpm (~13,400 ×
g) for 10 minutes at 4°C. Transfer the supernatant to a fresh
micro-centrifuge tube.
Note: When preparing samples with high content of fat,
proteins, polysaccharides, or extracellular material (e.g.,
muscle, fat tissue, or tuberous plant material), an additional
centrifugation may be required to remove insoluble material
from the samples. RNA remains in the upper aqueous phase
after centrifugation. However, when dealing with fat tissue,
the upper phase is a lipid layer that should be discarded.
Retain the clean homogenizing part for next step.
4. Add 200 µl of chloroform per 1 ml Buffer RZ used for
homogenization. Cap the tube securely and vortex for 15 s.
Incubate for 3 minutes at room temperature.
Note: If vortexing is not applicable, shake tube vigorously by
hand for 2 min.
5. Centrifuge the sample for 10 min at 12,000 rpm (~13,400 × g)
at 4°C. The mixture separates into a lower yellow phenol-
chloroform phase, an interphase, and a colorless upper
aqueous phase. RNA remains exclusively in the aqueous phase.
Pipette the aqueous phase out into a new tube.
6. Add the 0.5 volume ethanol (96%-100%) to the aqueous phase.
Mix thoroughly (participate may appear in this step). Transfer

the sample, including any precipitate that may have formed,
to an RNase-Free Spin Column CR3 placed in a 2 ml RNase-
Free Collection Tube. Close the lid gently, and centrifuge at
12,000 rpm (~13,400 × g) for 30 s at 4°C. Discard the flow-
through.
Note: If the sample is more than 700 µl, transfer the sample
to CR3 in two times and centrifuge separately.
7. Add 500 μl Buffer RD to the RNase-Free Spin Column CR3
(Ensure ethanol has been added). Close the lid gently, and
centrifuge at 12,000 rpm (~13,400 × g) for 30 s at 4°C. Discard
the flow-through.
8. Add 700 μl Buffer RW to the RNase-Free Spin Column CR3
(Ensure ethanol has been added). Close the lid gently, and
centrifuge at 12,000 rpm (~13,400 × g) for 30 s at 4°C. Discard
the flow-through.
9. Add 500 μl Buffer RW to the RNase-Free Spin Column CR3.
Close the lid gently, and centrifuge at 12,000 rpm (~13,400 × g)
for 30 s at 4°C. Discard the flow-through.
10. Set the RNase-Free Spin Column CR3 back to the Collection
Tube. Centrifuge at 12,000 rpm (~13,400 × g) for 2 min at 4°C
to dry the spin column membrane.
Note: The long centrifugation dries the spin column
membrane, ensuring that no ethanol is carried over during
RNA elution. Residual ethanol may interfere with
downstream reactions.
11. Place the RNase-Free Spin Column CR3 in a new 1.5 ml RNase-
Free Collection Tube (supplied). Add 30-100 μl RNase-Free
ddH2O directly to the spin column membrane. Close the lid
gently, incubate at room temperature (15–25°C) for 2 min.
Centrifuge at 12,000 rpm (~13,400 × g) for 2 min at 4°C to
elute the RNA.

Note: The volume of elution buffer should not be less than
30 μl, or it may affect recovery efficiency. To obtain higher
productivity, add the solution gained from step 11 to the
center of membrane again, let the columns stand for 1 min,
and then centrifuge.
Purified RNA should be stored at –70°C.
Ordering Information
RNA Isolation
Product Size Cat. no.
RNAstore Reagent 20 ml DP408-01
100 ml DP408-02
Reverse Transcription
Quantscript RT Kit 20 μl × 25 rxn KR103-03
20 μl × 100 rxn KR103-04
Real–Time PCR
SuperReal PreMix (SYBR Green) 50 μl × 50 rxn FP204-01
50 μl × 200 rxn FP204-02
Quant One Step qRT-PCR (SYBR Green) Kit 50 μl × 50 rxn FP303-01

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RNAsimple Total

RNAsimple Total RNA Kit

RNAsimple Total RNA Kit Handbook | 1

Notes of preventing RNase contamination

RNAsimple Total RNA Kit Handbook | 2

RNAsimple Total RNA Kit Handbook | 3

RNAsimple Total RNA Kit Handbook | 4

RNAsimple Total RNA Kit Handbook | 5

Product Size Cat. no.

RNAstore Reagent 20 ml DP408-01

Product Size Cat. no.

Quantscript RT Kit 20 μl × 25 rxn KR103-03

20 μl × 100 rxn KR103-04

Product Size Cat. no.

SuperReal PreMix (SYBR Green) 50 μl × 50 rxn FP204-01

50 μl × 200 rxn FP204-02

Quant One Step qRT-PCR (SYBR Green) Kit 50 μl × 50 rxn FP303-01

RNAsimple Total RNA Kit Handbook | 6

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