RNA Subcellular Isolation Kit(version A2)
Catalog No. 25501

Copyright 2014 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
any kind, either explicit or implied. Information may be changed or updated in this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2015 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
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All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Flow Chart of Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
RNA Subcellular Isolation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Section A: Preparation of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Section B: Preparation of Fresh Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Section C: Preparation of Frozen Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section D: Nuclear & Cytoplasmic RNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section E: Total RNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section F: RNA Quantification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section G: Downstream Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section H: Data Normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Appendix
Section K. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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Overview
The RNA Subcellular Isolation Kit offers an efficient method to isolate RNA from different cellular
fractions using cultured cells or fresh tissues. Unlike most RNA preparation methods that isolate
only total RNA, Active Motif’s RNA Subcellular Isolation Kit enables purification of large and small
RNAs from both nuclear and cytoplasmic fractions without cross contamination or the use ofphenolic compounds. Additionally, total RNA can be isolated from whole cells and frozen tissue.
Understanding the transcriptome is a complex process. RNA molecules are present at different
levels of maturation and processing within a cell at any given time, which can complicate andbias downstream analysis. In some instances, it may be advantageous to identify splice junctions
and measure gene expression levels without the background of intronic RNAs from the nucleus.
Conversely, it may be desirable to study chromatin-associated RNA or nuclear RNA maturation
without the influence of mature RNAs from the cytoplasm. Finally, given the diversity of ncRNAs,
understanding RNA localization may help to inform about RNA function. Active Motif’s RNA
Subcellular Isolation Kit provides a solution to these challenges by enabling the isolation of RNA
from specific cellular fractions and making it easier to analyze the RNA of interest or to determine
RNA localization.
The RNA Subcellular Isolation method works by lysing cells or tissue samples in complete lysis
buffer. The lysate is then separated by centrifugation, with the supernatant containing cytoplasmic RNA and the pellet containing nuclear RNA. A guanidine-based buffer and ethanol are added
to each RNA fraction before being loaded onto spin-columns. The column binds the RNA allowing
any contaminating protein to flow through the membrane or remain unbound. Following a wash
step, the RNA fractions are eluted and quantified. The purified RNA is suitable for use in a variety
of downstream applications, including reverse transcription PCR (RT-qPCR), Northern blotting andRNA-seq.
The RNA Subcellular Isolation Kit includes enough reagents to perform 15 cytoplasmic and
15 nuclear preparations, or 30 total RNA purifications.

product

formatcatalog no.

RNA Subcellular Isolation Kit

30 rxns

25501

DNase I Treatment Kit

30 rxns

25502

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1Flow Chart of Process

Subcellular Isolation

Total RNA

Cultured Cells or Fresh Tissue

Cultured Cells, Fresh or Frozen Tissue

Add Lysis Buffer
Incubate on ice 10-20 min.
Centrifuge toseparate cell fractions

Supernatant containing
cytoplasmic RNA

Pellet containing
nuclear RNA

Add Buffer G
Add 70% Ethanol

Add Buffer G
Add 70% Ethanol

Add Buffer G
Add 70% Ethanol

Load Spin Column

Load Spin Column

Load Spin Column

Wash

Wash

Wash

Elute

Elute

Elute

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Kit Performance and Benefits
The RNA Subcellular Isolation Kit is designed to isolate nuclear and cytoplasmic RNA from cultured cells and fresh tissue, or total RNA from cultured cells and frozen tissue.
Maximum Column Binding Capacity

40 µg

Maximum Column Loading
Volume750 µlSize of purified RNA

RNA between the sizes of 75 nt to 7,000 nt

Maximum Amount of Starting
Material
Mammalian Cells
Mammalian Tissues

4 x 106 cells (Cytoplasmic and Nuclear RNA)2 x 106 cells (Total RNA)
15 mg

Assay Time

2 hours

Average Yields
HeLa Cytoplasmic RNA
HeLa Nuclear RNA
HeLa Total RNA

See page 7 for more guidance on cell numbers and RNA
yields~12-25 µg at 250-500 ng/µl from 2 x 106 cells~12-25 µg at 250-500 ng/µl from 2 x 106 cells~25-45 µg at 500-900 ng/µl from 2 x 106 cells

RNA Subcellular Isolation Kit

Figure 1: Agarose gel of nuclear and cytoplasmic RNA fractions isolated with the RNA Subcellular Isolation Kit.
RNA was isolated as nuclear and cytoplasmic fractions using the RNA Subcellular Isolation Kit. Following purification, half ofthe nuclear RNA was treated with DNase I to remove genomic DNA contamination using the DNase I Treatment Kit (Active
Motif Catalog No. 25502). 1 µg of each RNA fraction was loaded onto a 1.5% agarose gel for analysis..
Lanes M = 100 bp DNA ladder
Lanes 1-2 = Cytoplasmic RNA
Lanes 3-4 = Nuclear RNA before DNase I treatment

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Lanes 5-6 = Nuclear RNA after DNase I treatment

Figure 2: Cytoplasmic RNA trace pre- and post ribosomal RNA (rRNA) depletion.
Cytoplasmic RNA was isolated from HeLa cells using the RNA Subcellular Isolation Kit. TapeStation analysis was performed
to analyze the recovered RNA before and after rRNA depletion. The yellow and green traces represent two cytoplasmic
samples pre-rRNA depletion. The two large peaks represent the 18S and 28S rRNA. The blue and red traces represent thesame samples after rRNA depletion. The 18S and 28S peaks are no longer present. The size of the purified RNA spans from
75 nucleotides to 7,000 nucleotides.

Percent of Total RNA

Expression analysis reveals RNA localization
100
90
80
70
60
50
40
30
20
10
0

NuclearCytoplasmic

MALAT1

NEAT1

TUG1

S14

BIRC5

Figure 3: Subcellular RNA isolation to identify RNA localization.
Nuclear, cytoplasmic and total RNA were isolated from HeLa cells using the RNA Subcellular Isolation Kit. Following purification and DNase I treatment of the nuclear and total RNA fraction with Active Motif’s DNase I Treatment Kit (Catalog No.
25502) purified RNA was subjected to reverse transcription and qPCR. Each subcellular fraction was plotted as a percentage
of total RNA. The data shows the localization of various RNAs. MALAT1, NEAT1 and TUG1 are lncRNAs. Their expression analysis data is consistent with published RNA FISH data for the same RNAs and cell type. Both S14, which is a ribosomal RNA, andBIRC5, a mRNA, are expected to localize within the cytoplasm as demonstrated above.

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4RNA Subcellular Isolation Kit Components and Storage
RNA Subcellular Isolation Kits are for research use only. Not for use in diagnostic procedures. Allcomponents are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

1 M DTT

100 µl-20°C

10X PBS

120 ml

4°C

Lysis Buffer AM7

5 ml

4°C

Buffer G

20 ml

4°C

Purification Columns

30 ea

4°C

DEPC Water

10 ml

RT

Additional materials required• Conical tubes and centrifuge• 70% Ethanol• β-mercaptoethanol• RNase-free microcentrifuge tubes and microcentrifuge• Vortex set to highest setting• Ice bucket and ice• Nanodrop, Qubit or similar device for RNA quantification• Sample material for RNA analysis (cultured cells, fresh or frozen tissue)• Trypsin (cultured cell samples)• Hemacytometer (cultured cell samples)• Microscope (cultured cell samples)• Razor blades (fresh and frozen tissue samples)• Analytical balance (fresh and frozen tissue samples)• Syringes with small (27-29 gauge), medium (21-23 gauge) and large (15-17 gauge)
gauge needles (fresh tissue samples)• Mortar and pestle (frozen tissue samples)• Liquid nitrogen (frozen tissue samples)• Dry ice (frozen tissue samples)• (Optional) DNase I Treatment Kit (Active Motif, Catalog No. 25502)

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Protocols
Buffer Preparation and Recommendations
10X PBS
Prepare a 1X PBS by diluting the 10X PBS in sterile water. For every 10 ml needed, dilute 1 ml 10X
PBS into 9 ml sterile water.
Preparation of Complete Lysis Buffer
Prepare the amount of Complete Lysis Buffer required for the assay by adding 5 µl of the provided 1 M DTT per 1 ml Lysis Buffer AM7 (see the Quick Chart for Preparing Buffers in this section).
Mix well by pipetting. After use, discard remaining Complete Lysis Buffer.
Preparation of Complete Buffer G
Buffer G contains salts that will precipitate out of solution during cold temperature storage. We
recommend placing the buffer at room temperature 1 hour prior to use to allow the solution towarm up. Ensure that the salts are fully dissolved into solution before use by vortexing at room
temperature. Prepare the amount of Complete Buffer G required for the assay. Transfer the necessary amount of Buffer G to a clean container. Add 10 µl of β-mercaptoethanol per 1 ml Buffer G(see the Quick Chart for Preparing Buffers in this section). Mix well by vortexing. Keep the buffer
at room temperature during the assay. After use, discard remaining Complete Buffer G.
Preparation of Wash Buffer
Prepare the amount of Wash Buffer required for the assay. Transfer the necessary amount of Buffer G (without β-mercaptoethanol) for the wash steps into a clean container. Add an equal volumeof 70% ethanol (e.g. Add 5 ml 70% ethanol to 5 ml Buffer G). Invert repeatedly. The buffer can be
stored at room temperature after the addition of ethanol.

Quick Chart for Preparing Buffers
Total RNAFor 1 rxn

Nuc & Cyto RNA
For 1 rxn each

Total, Nuc & Cyto RNA
For 1 rxn each

1 M DTT
Lysis Buffer AM7
TOTAL REQUIRED–––

0.6 µl120 µl120 µl0.6 µl120 µl120 µlComplete Buffer G

β-mercaptoethanol
Buffer G
TOTAL REQUIRED

3.5 µl346.5 µl
350 µl6 µl594 µl600 µl10 µl990 µl
1 ml

Wash Buffer

70% Ethanol
Buffer G
TOTAL REQUIRED

300 µl300 µl600 µl600 µl600 µl1.2 ml

1 ml
1 ml
2 ml

70% Ethanol

TOTAL REQUIRED

1.25 ml

2.65 ml

4 ml

DEPC Water

TOTAL REQUIRED

50 µl

100 µl150 µlReagents to prepare

Components

Complete Lysis Bufferwww.activemotif.com

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Assay Protocol
Read the entire protocol before use. Ensure work space and lab supplies are free from RNases. Wear gloves at all times.
The RNA Subcellular Isolation Kit is designed to purify nuclear and cytoplasmic RNA from cultured
cells and fresh tissue, or to isolate total RNA from whole cells, fresh or frozen tissue. Isolated
RNA can be quantified by Nanodrop, Qubit or equivalent device for RNA quantification. For data
analysis requiring normalization, it may be beneficial to set up duplicate reactions. Use one reaction to prepare nuclear and cytoplasmic RNA and the second reaction to prepare total RNA fromthe same sample material. For details on various normalization options, please refer to Appendix
Section H on page 13.
RNA purified using the RNA Subcellular Isolation Kit is suitable for use in a variety of downstream
applications including RT-qPCR, Northern blot and RNA-seq. Additional DNase treatment can be
performed on the purified RNA using Active Motif’s DNase I Treatment Kit (Catalog No. 25502).
Determine the amount of starting material necessary to isolate sufficient RNA yields for downstream analysis by taking into consideration that ribosomal RNA (rRNA) accounts for a majority(~80%) of the total RNA in a cell. If you plan to perform rRNA depletion or poly adenylation selection following RNA isolation, you may need to increase the amount of starting material. Somegeneral guidelines of RNA yields are provided below for reference.
Cell
NumberCytoplasmic
RNA Yield

NuclearRNA Yield

Total
RNA Yield

2 Million

20 µg

20 µg

40 µg

1 Million

10 µg

10 µg

30 µg

500,000

5 µg

5 µg

15 µg

250,000

3 µg

3 µg

8 µg

100,000

1.5 µg1.5 µg

3 µg

Section A: Preparation of Cultured Cells
The protocol below describes the preparation of one 150 mm plate. Each 150 mm plate should
generate enough cells to prepare multiple RNA isolation reactions.
1.

Grow cells to 90% confluence in a 150 mm plate. Treat cells if desired.

2.

Wash cells once with 10 ml 1X PBS

3.

Add 4 ml of Trypsin to each plate. Incubate at 37°C for 5 minutes.

4.

Add 5 ml of complete media to each plate and pipet up and down to detach the cells fromthe plate. Transfer cells to a 15 ml conical tube.

5.

Centrifuge for 5 minutes at 450 x g.

6.

Remove the supernatant and resuspend the cells in 10 ml 1X PBS.
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7.

Count the cells using a hemacytometer and Trypan blue.

8.

Set up reactions by adding desired number of cells to a microcentrifuge tube. If both subcellular fractionated and total RNA are needed, prepare two tubes for each sample. See tableon page 7 for guidelines of RNA yields to determine the appropriate number of cells neededfor downstream analysis. Discard any remaining cells.

9.

Centrifuge the culture cell samples at 14,000 rpm for 5 minutes. Discard the supernatant.

10. Proceed to Section D for Nuclear & Cytoplasmic RNA Isolation. Proceed to Section E for
Total RNA Isolation.

Section B: Preparation of Fresh Tissue1.

Add ice-cold 1X PBS to a conical of the appropriate size for the tissue of interest until theconical tube is 3/4 filled with PBS.

2.

Place conical tube with PBS on a balance and tare the value to zero.

3.

Working quickly, isolate your tissue of interest. Immediately wash with 10 ml ice-cold 1X PBS
and add tissue to the tared conical tube. Record the mass of the tissue. Keep the tissue in
PBS until ready to use. If preparing multiple samples, place tube on ice until all samples areready.

4.

We recommend working with 5 -15 mg of tissue per RNA isolation in microcentrifuge tubes.
Based on the total mass of the initial tissue, prepare the appropriate number of microcentrifuge tubes. Add 1 ml ice-cold 1X PBS to each microcentrifuge tube.
Note:

Any unused tissue can be transferred in 5-15 mg quantities to a cryo vial (without PBS). Flash freeze the cryo vial in liquid nitrogen and immediately store at-80°C for future use.

5.

Transfer the tissue in PBS to an empty 150 mm cell culture plate. Use a sterile razor blade todice the tissue into small pieces (~2 mm cubes).

6.

Place a single microcentrifuge tube with 1 ml PBS on a balance and tare the value to zero.
Transfer a small tissue section into the microcentrifuge containing the PBS. Continue until
the desired mass is reached. Record the mass of the tissue. Place the tube back on ice.

7.

Repeat step 6 until all of the tissue has been transferred to microcentrifuge tubes.

8.

Keep the tissue samples to be processed on ice.

9.

Shear the fresh tissue samples using a syringe and a large gauge needle (15-17). Continue
to shear until the tissue is broken into smaller pieces.

10. Repeat shearing using a syringe and a medium gauge needle (21-23). Continue to break
down the tissue into smaller pieces.
11.

Repeat shearing using a syringe and a small gauge needle (27-29). The tissue should be ahomogenous solution.

12. Place the sample on ice.
13. Repeat Steps 9-12 for each additional tissue sample until all samples have been processed.
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814. Centrifuge the fresh tissue samples at 14,000 rpm for 5 minutes. Discard the supernatant.
15. Proceed to Section D for Nuclear & Cytoplasmic RNA Isolation. Proceed to Section E for
Total RNA Isolation.

Section C: Preparation of Frozen Tissue
1.

Use frozen tissue that has been flash frozen immediately after excision and stored at -80°C
for no more than one month.

2.

Chill a clean mortar and pestle that is free from RNase contamination.

3.

Remove your frozen tissue sample from -80°C and place on dry ice.

4.

We recommend working with 5 -15 mg of tissue per RNA isolation. If necessary, transfer thetissue to an empty 150 mm cell culture plate on dry ice. Working quickly, use a razor blade
to dice the tissue into small pieces (~2 mm cubes). Do not allow the tissue to thaw. Anyunused tissue should be stored at -80°C.

5.

Transfer the desired amount of tissue to the chilled mortar and pestle and cover with liquid
nitrogen. Use the pestle to grind the tissue into a fine powder. Collect the ground tissue into
the center of the mortar using a sterile pipette tip.
Note:

6.

If preparing multiple reactions from the same tissue sample, the tissue can beprepared in bulk and then transferred to individual tubes.

Add 350 µl Complete Buffer G per reaction to the mortar to dissolve the tissue powder.
Note:

For multiple reactions, scale up the amount of Complete Buffer G needed(e.g. Two reactions with 30 mg of tissue would use 700 µl Complete Buffer G).

7.

Allow the mortar to warm up enough to transfer the dissolved tissue into a microcentrifuge
tube. Add 350 µl per tube if working with multiple reactions. Place the samples in an ice
bucket with ice until all samples have been processed.

8.

Repeat steps 2-7 if working with multiple tissue samples, cleaning the mortar and pestle inbetween each sample.

9.

Proceed to Section E, Step 2 to continue with total RNA isolation.

Section D: Nuclear & Cytoplasmic RNA Isolation
1.

Resuspend each cell pellet in 120 µl Complete Lysis Buffer.

2.

Incubate samples on ice: Cultured cells: Incubate for 10 minutes on ice.
Tissue samples: Incubate for 20 minutes on ice.

3.

Centrifuge at 14,000 rpm for 5 minutes at 4°C.

4.

Prepare a new RNase-free microcentrifuge tube for each sample and label as Cytoplasmic.
Transfer the supernatant from Step 3 to the new tube. Take care to remove all of the supernatant without disturbing the pellet.
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5.

The remaining pellet contains the nuclear fraction. Label the tubes as Nuclear. Wash thenuclear pellets by adding 150 µl of 70% ethanol to each tube. Centrifuge at 14,000 rpm for 5
minutes at 4°C. Carefully remove and discard supernatant.

6.

Add Complete Buffer G to each tube as follows:
Cytoplasmic: Add 250 µl Complete Buffer G
Nuclear: Add 350 µl Complete Buffer G.

7.

Vortex all tubes on highest setting for 30 seconds.

8.

Add 350 µl of 70% ethanol to all tubes.

9.

Using a pipette and P200 tip, pipette up and down to homogenize the solution. (The solution is very gelatinous at first, but this should reduce with pipetting.)

10. Prepare separate purification columns for each cytoplasmic and nuclear sample. Add theentire sample to its corresponding purification column.
11.

Centrifuge the columns with their collection tubes at 14,000 rpm for 1 minute at 4°C. Discard
the flow through.

12. Add 600 µl Wash Buffer to each column.
13. Centrifuge the columns with their collection tubes at 14,000 rpm for 1 minute at 4°C. Discard
the flow through.
14. Add 600 µl of 70% ethanol to each column.
15. Centrifuge the columns with their collection tubes at 14,000 rpm for 1 minute at 4°C. Discard
the flow through.
16. Repeat centrifugation at 14,000 rpm for 2 minute at 4°C to remove any remaining liquid.
17.

Transfer each column to a new RNase-free microcentrifuge tube. Make sure the new collection tubes are labeled for each sample.

18. Add 50 µl DEPC water to the center of each column. Let stand for 1 minute.
19. Centrifuge the columns at 14,000 rpm for 1 minute at 4°C. Eluate contains the Subcellular
RNA fractions.
20. Discard the columns. Place the nuclear and cytoplasmic RNA samples on ice.
21. Proceed to Section F for RNA quantification.

Section E: Total RNA Isolation
1.

Resuspend each cell pellet in 350 µl Complete Buffer G.

2.

Vortex tubes on highest setting for 30 seconds.

3.

Add 350 µl of 70% ethanol to each tube.

4.

Using a pipette and P200 tip, pipette up and down to homogenize the solution. (The solution is very gelatinous at first, but this should reduce with pipetting.).

5.

Prepare separate purification columns for each sample. Add the entire sample to its corresponding purification column.
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6.

Centrifuge the columns with their collection tubes at 14,000 rpm for 1 minute at 4°C. Discard
the flow through.

7.

Add 600 µl Wash Buffer to each column.

8.

Centrifuge the columns with their collection tubes at 14,000 rpm for 1 minute at 4°C. Discard
the flow through.

9.

Add 600 µl of 70% ethanol to each column.

10. Centrifuge the columns with their collection tubes at 14,000 rpm for 1 minute at 4°C. Discard
the flow through.
11.

Repeat centrifugation at 14,000 rpm for 2 minute at 4°C to remove any remaining liquid.

12. Transfer each column to a new RNase-free microcentrifuge tube. Make sure the new collection tubes are labeled for each sample.
13. Add 50 µl DEPC water to the center of each column. Let stand for 1 minute.
14. Centrifuge the columns at 14,000 rpm for 1 minute at 4°C. Eluate contains the total RNA.
15. Discard the columns. Place total RNA samples on ice.
16. Proceed to Section F for RNA quantification.

Section F: RNA Quantification
1.

Quantify RNA using the absorbance 260nm value with a Nanodrop or equivalent spectrophotometric device. If anticipating low RNA yield, use a fluorescent RNA detection method,such as Qubit, to quantify the RNA samples. For accurate quantification it is important thatthe sample measurement falls within the detectable range of the instrument used.

2.

We recommend preparing aliquots of the RNA and storing at -20°C to -80°C. It is important
to avoid freeze/thaw cycles when working with RNA.

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Appendix
Section G. Downstream Applications
Purified RNA is suitable for use in a variety of downstream applications, including reverse transcription PCR (RT-qPCR), Northern blotting and RNA-seq.
DNase I Treatment
If desired, RNA samples can be treated with DNase I to ensure the removal of genomic DNA fromthe nuclear or total RNA fractions. It is recommended to use Active Motif’s DNase I Treatment Kit(Catalog No. 25502) or equivalent DNase treatment protocol. Following DNase treatment, theRNA sample should be quantified using the methods described in Section F.
RT-qPCRFor RT-qPCR we recommend the use of a two-step real time PCR in which the reverse transcription reaction is performed separately from the real-time PCR assay. Commercially available reverse transcription kits (e.g. iScript™ cDNA Synthesis Kit, Bio-Rad Cat No. 1708890) can be used togenerate cDNA. Follow the manufacturer’s recommendations for reverse transcription and qPCR.
Section H below discusses considerations for control reactions and data normalization.
RNA-Seq
For RNA-seq, we recommend checking with your sequencing facility to determine their requirements for RNA material. Many sequencing facilities will perform poly adenylation selection
or rRNA depletion as part of the library preparation process. The sequencing facility will also
provide guidelines on the amount of RNA required.

Figure 4: RNA-Seq data using RNA fractions isolated with the RNA Subcellular Isolation Kit.
Nuclear, cytoplasmic and total RNA were isolated from HeLa cells using the RNA Subcellular Isolation Kit. RNA was
subjected to ribosomal RNA depletion during NGS library preparation. Samples were then sequenced using the Illumina
HiSeq and 100 bp paired end reads with 50 M reads per sample. TUG1 is a lncRNA known to have both nuclear and cytoplasmic localizations.

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12Section H. Data Normalization
Gene expression analysis using real time reverse transcription quantitative PCR (RT-qPCR) is often
the method of choice for accurate expression profiling of select genes. This method is highly sensitive, reproducible and has a large dynamic range. However, it is important to consider proper
controls for gene normalization to account for differences in starting material, RT efficiency, RNAintegrity and RNA localization (if evaluating different subcellular fractions). We will present two
approaches for data normalization below. Determine the appropriate method for your researchanalysis.
Normalization using a reference gene
Data normalization can be performed by including an endogenous control in the assay to correct
for sample-to-sample variations. This control is usually a reference gene that is constitutively
expressed (also referred to as a housekeeping gene). Individual RT-qPCR samples are normalized
to the reference gene as a fold-change to allow for direct comparison between different samples.
The challenge with this approach is to select the appropriate control gene. The gene should notshow variation in expression in the tissues or cells being investigated, nor should it change in
response to experimental treatments. Another consideration when evaluating RNA samples fromdifferent subcellular fractions is the localization of the control gene. If the control gene is localized to one subcellular fraction, use of this gene for analysis with a different subcellular fraction
can bias the results. Therefore, it is often necessary to use several reference genes.
Fold change = 2^ (Ct control gene – Ct test gene)

Normalization using total RNA content
Data normalization is also performed against total cellular RNA. This method requires accuratequantification of the total RNA fraction. RNA extracted from nuclear and cytoplasmic fractions canbe compared to the total RNA levels. By using total RNA for normalization, bias that is the resultof RNA localization is avoided. However, it does require the preparation of nuclear, cytoplasmic
and total RNA from the same sample material.
% of Input = 100 x [2^ (Ct total RNA – Ct RNA fraction)www.activemotif.com

13Section K: Troubleshooting Guide
Problem/question

Recommendation

Little or no recovery of RNA

Make sure to use RNase-free tubes, tips and reagents when working with RNA samples.
Wear gloves at all times when handling the samples.
Quantify RNA using a spectrophotometer or fluorescent detection method. For accuratequantification it is important that the sample measurement falls within the detectable rangeof the instrument used.
Quantification of RNA may be influenced by the presence of genomic DNA in the sample. If
desired, perform a DNase treatment of the isolated RNA. Expect to see a reduction in theyield following DNase treatment.

I see genomic DNA in mysampleThe RNA Subcellular Isolation Kit can be used to isolate RNA from nuclear and cytoplasmic
fractions and total RNA. It is normal to see genomic DNA isolated in the nuclear and total
RNA samples. To remove genomic DNA from the RNA samples, we recommend using Active
Motif’s DNase I Treatment Kit (Catalog No. 25502).

Is it normal for my sample
consistency to change after
addition of 70% ethanol?

Yes, the combination of the Complete Buffer G and the 70% ethanol will change theconsistency of the sample. Use a P200 pipette and tip and mix the sample by pipetting up
and down 15-20 times. The sample consistency should become easier to work with after
pipetting. If the sample continues to be difficult to work with, use a 27-29 gauge needle
and syringe to beak up the material.

Can I elute in less volume?

We recommend elution from the purification columns with 50 µl DEPC water. Eluting insmaller volumes significantly reduces the amount of RNA recovered. If necessary, RNA canbe concentrated with additional purification steps following elution off the column.

What species can I use thiskit for?

The RNA Subcellular Isolation Kit has been used to enrich for RNA from mammaliansamples. We have not tested with other species.

Storage of RNA

RNA samples may be stored at -20°C to -80°C. We recommend preparing smaller aliquotsof RNA to avoid freeze/thaw cycles.

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Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
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Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

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