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The BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ Complete Dicer RNAi Kit facilitate generation of purified diced siRNA duplexes (d-siRNA) that are suitable for use in RNAi analysis of a target gene in mammalian cells. The kits contain the BLOCK-iT™ Dicer Enzyme for dicing dsRNA, reagents to purify the d-siRNA, and an optimized transfection reagent for highly efficient delivery of d-siRNA to mammalian cells.
Note: The BLOCK-iT™ Complete Dicer RNAi Kit also includes the BLOCK-iT™ RNAi TOPO® Transcription Kit to facilitate high-yield generation of purified dsRNA. For more information, refer to the BLOCK-iT™ RNAi TOPO® Transcription Kit manual. This manual is supplied with the BLOCK-iT™ Complete Dicer RNAi Kit, but is also available for downloading from www.invitrogen.com or by contacting Technical Service (page 30).
Advantages of the BLOCK-iT™ Dicer RNAi Transfection Kit
Using the BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ Complete Dicer RNAi Kit to generate d-siRNA for RNAi analysis in mammalian provides the following advantages:
Purpose of this Protocol
The RNAi Pathway and How Dicer Works
The RNAi Pathway
RNAi describes the phenomenon by which dsRNA induces potent and specific inhibition of eukaryotic gene expression via the degradation of complementary messenger RNA (mRNA), and is functionally similar to the processes of post-transcriptional gene silencing (PTGS) or cosuppression in plants (Cogoni et al., 1994; Napoli et al., 1990; Smith et al., 1990; van der Krol et al., 1990) and quelling in fungi (Cogoni and Macino, 1999; Cogoni and Macino, 1997; Romano and Macino, 1992). In plants, the PTGS response is thought to occur as a natural defense against viral infection or transposon insertion (Anandalakshmi et al., 1998; Jones et al., 1998; Li and Ding, 2001; Voinnet et al., 1999).
In eukaryotic organisms, dsRNA produced in vivo or introduced by pathogens is processed into 21-23 nucleotide double-stranded short interfering RNA duplexes (siRNA) by an enzyme called Dicer, a member of the RNase III family of double-stranded RNA-specific endonucleases (Bernstein et al., 2001; Ketting et al., 2001). Each siRNA then incorporates into an RNA-induced silencing complex (RISC), an enzyme complex that serves to target cellular transcripts complementary to the siRNA for specific cleavage and degradation (Hammond et al., 2000; Nykanen et al., 2001).
For more information about the RNAi pathway and the mechanism of gene silencing, refer to recent reviews (Bosher and Labouesse, 2000; Hannon, 2002; Plasterk and Ketting, 2000; Zamore, 2001).
Performing RNAi Analysis in Mammalian Cells
A number of kits including the BLOCK-iT™ RNAi TOPO® Transcription Kit now exist to facilitate in vitro production of dsRNA that is targeted to a particular gene of interest. The dsRNA may be introduced directly into some invertebrate organisms or cell lines, where it functions to trigger the endogenous RNAi pathway resulting in inhibition of the target gene. Long dsRNA duplexes cannot be used directly for RNAi analysis in most somatic mammalian cell lines because introduction of long dsRNA into these cell lines induces a non-specific, interferon-mediated response, resulting in shutdown of translation and initiation of cellular apoptosis (Kaufman, 1999). To avoid triggering the interferon-mediated host cell response, dsRNA duplexes of less than 30 nucleotides must be introduced into cells (Stark et al., 1998). For optimal results in gene knockdown studies, the size of the dsRNA duplexes (i.e. siRNA) introduced into mammalian cells is further limited to 21-23 nucleotides.
Using the Kit for RNAi Analysis
The BLOCK-iT™ Dicer RNAi Transfection Kit and the BLOCK-iT™ Complete Dicer RNAi Kit facilitate in vitro production of a complex pool of 21-23 nucleotide siRNA duplexes that is targeted to a particular gene of interest. The kits use a recombinant human Dicer enzyme (see below for more information) to cleave a long dsRNA substrate (produced with the BLOCK-iT™ RNAi TOPO® Transcription Kit) into a pool of 21-23 nucleotide d-siRNA that may be transfected into mammalian cells. Introduction of d-siRNA into the cells then triggers the endogenous RNAi pathway, resulting in inhibition of the target gene. For a diagram of the process, see the figure below.
BLOCK-iT™ Dicer Enzyme
BLOCK-iT™ Dicer is a recombinant human enzyme (Myers et al., 2003; Provost et al., 2002) that cleaves long dsRNA processively into 21-23 nucleotide d-siRNA duplexes with 2 nucleotide 3' overhangs. The Dicer enzyme is a member of the RNase III family of double-stranded RNA-specific endonucleases, and consists of an ATP-dependent RNA helicase domain, a Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains, and a dsRNA-binding domain (Bernstein et al., 2001; Zamore, 2001). In addition to its role in the generation of siRNA, Dicer is also involved in the processing of short temporal RNA (stRNA) (Hutvagner et al., 2001; Ketting et al., 2001) and microRNA (miRNA) (Carrington and Ambros, 2003) from stable hairpin or stem-loop precursors.
Shipping/Storage
The BLOCK-iT™ Dicer RNAi Kits are shipped as described below. Upon receipt, store each item as detailed below. For more detailed information about the reagents supplied with the BLOCK-iT™ RNAi TOPO® Transcription Kit, refer to the BLOCK-iT™ RNAi TOPO® Transcription Kit manual.
Box | Component | Shipping | Storage |
---|---|---|---|
1 | BLOCK-iT™ Dicer Enzyme Kit | Dry ice | -20 ° C |
2 | BLOCK-iT™ RNAi Purification Kit | Room temperature | Room temperature |
3 | Lipofectamine® 2000 Reagent | Blue ice | +4 ° C (do not freeze) |
4 - 6 | BLOCK-iT™ RNAi TOPO® Transcription Kit | BLOCK-iT™ TOPO® Linker Kit and BLOCK-iT™ RNAi Transcription Kit: Dry ice BLOCK-iT™ RNAi Purification Kit: Room temperature | BLOCK-iT™ TOPO® Linker Kit and BLOCK-iT™ RNAi Transcription Kit: -20 ° C BLOCK-iT™ RNAi Purification Kit: Room temperature |
BLOCK-iT™ Dicer Enzyme Kit
The following reagents are included with the BLOCK-iT™ Dicer Enzyme Kit (Box 1). Store the reagents at -20 ° C.
Reagent | Composition | Amount |
BLOCK-iT
™ Dicer Enzyme
|
1 U/µl in a proprietary buffer
|
300 µl
|
10X Dicer Buffer
|
Proprietary
|
150 µl
|
50X Dicer Stop Buffer
|
0.5 mM EDTA, pH 8.0
|
30 µl
|
RNase-Free Water
|
--
|
1.5 ml
|
Unit Definition
One unit of BLOCK-iT™ Dicer enzyme cleaves 1 µg of double-stranded RNA (dsRNA) in 16 hours at 37 ° C.
BLOCK-iT™ RNAi Purification Kit
The following reagents are included with the BLOCK-iT™ RNAi Purification Kit (Box 2). Store reagents at room temperature. Use caution when handling the RNA Binding Buffer. (see the next page for more information)
Note: Catalog no. K3650-01 includes two boxes of BLOCK-iT™ RNAi Purification reagents. One box is supplied with the BLOCK-iT™ RNAi TOPO® Transcription Kit for purification of sense and antisense single-stranded RNA (ssRNA). The second box is supplied for purification of diced siRNA (d-siRNA).
Reagent | Composition | Amount |
RNA Binding Buffer
|
Proprietary
|
1.8 ml
|
5X RNA Wash Buffer
|
Proprietary
|
2.5 ml
|
RNase-Free Water
|
--
|
800 µl
|
RNA Spin Cartridges
|
--
|
10
|
RNA Recovery Tubes
|
--
|
10
|
siRNA Collection Tubes*
|
--
|
5
|
50X RNA Annealing Buffer
|
500 mM Tris-HCl, pH 8.0
1 M NaCl
50 mM EDTA, pH 8.0
|
50 µl
|
*siRNA Collection Tubes are used for purification of dsiRNA only, and are not required for the purification of the ssRNA.
The RNA Binding Buffer supplied in the BLOCK-iT™ RNAi Purification Kit contains guanidine isothiocyanate. This chemical is harmful if it comes in contact with the skin or is inhaled or swallowed. Always wear a laboratory coat, disposable gloves, and goggles when handling solutions containing this chemical.
Do not add bleach or acidic solutions directly to solutions containing guanidine isothiocyanate or sample preparation waste. Guanidine isothiocyanate forms reactive compounds and toxic gases when mixed with bleach or acids.
Lipofectamine® 2000 Reagent
Each BLOCK-iT™ Dicer RNAi Kit includes Lipofectamine® 2000 Reagent (Box 3) for high efficiency transfection of d-siRNA into mammalian cells. Lipofectamine® 2000 Reagent is supplied as follows:
Size: 0.75 ml
Concentration: 1 mg/ml
Storage: +4 °C; do not freeze
BLOCK-iT™ RNAi TOPO® Transcription Kit
The BLOCK-iT™ Complete Dicer RNAi Kit (Catalog no. K3650-01) includes the BLOCK-iT™ RNAi TOPO® Transcription Kit to facilitate production of double-stranded RNA (dsRNA) from your gene of interest. Refer to the BLOCK-iT™ RNAi TOPO® Transcription Kit manual for a detailed description of the reagents provided with the kit and instructions to produce dsRNA.Accessory Products
Introduction
The products listed in this section may be used with the BLOCK-iT™ Dicer RNAi Kits. For more information, refer to our Web site (www.invitrogen.com) or call Technical Service.
Accessory Products
Some of the reagents supplied in the BLOCK-iT™ Dicer RNAi Kits as well as other products suitable for use with the kit are available separately from Invitrogen. Ordering information is provided below.
a. This balances the risk of including regions of strong homology between the target gene and other genes that could result in non-specific off-target effects during RNAi analysis with the benefits of using a more complex pool of siRNA.
b. When producing sense and antisense transcripts of the target template, the highest transcription efficiencies are obtained with transcripts in the 500 bp to 1 kb size range. Target templates outside this size range transcribe less efficiently, resulting in lower yields of dsRNA.
c. Double-stranded RNA that is under 1 kb in size is efficiently diced. Larger dsRNA substrates can be used but yields may decline as the size increases.
Introduction
Once you have produced your target dsRNA, you will perform an in vitro dicing reaction using the reagents supplied in the BLOCK-iT™ Dicer Enzyme Kit (Box 1) to generate d-siRNA duplexes of 21-23 nucleotides in size.
BLOCK-iT™ Dicer Enzyme Activity
One unit of BLOCK-iT™ Dicer Enzyme cleaves 1 µg of dsRNA in 16 hours at 37° C. Note that the Dicer enzyme does not cleave dsRNA to d-siRNA with 100% effi-ciency, i.e. dicing 1 µg of dsRNA does not generate 1 µg of d-siRNA. Under these optimal reaction conditions, the Dicer enzyme cleaves dsRNA to d-siRNA with an efficiency of approximately 25-35%. For example, dicing 60 µg of dsRNA in a 300 µl dicing reaction typically yields 12-18 µg of d-siRNA following purification.
Note the following:
Amount of dsRNA to Use
For a typical 300 µl dicing reaction, you will need 60 µg of target dsRNA. If you want to dice less than 60 µg of dsRNA, scale down the entire reaction proportionally.
The total volume of dsRNA added should not exceed half the volume of the reaction. Thus, for best results, make sure that the starting concentration of your dsRNA is US 400 ng/µl.
Positive Control
If you are using the BLOCK-iT™ Complete Dicer RNAi Kit, and have performed all of the recommended control reactions using the control reagents supplied in the BLOCK-iT™ RNAi TOPO® Transcription portion of the kit, you should have purified dsRNA representing a 1 kb portion of the lacZ gene. We recommend setting up a separate dicing and purification reaction using the control lacZ dsRNA. You can then co-transfect the resulting purified lacZ d-siRNA and the pcDNA™1.2/V5-GW/lacZ control plasmid supplied with the kit into your mammalian cell line as a positive control for the RNAi response in that cell line. Alternatively, you may use the lacZ d-siRNA as a negative control for non-specific, off-target effects in your cell line.
Steps to Avoid RNase Contamination
Materials Needed
Have the following reagents on hand before beginning:
Dicing Procedure
Follow the procedure below to perform the dicing reaction. Make sure that the volume of dsRNA added does not exceed half the volume of the reaction (i.e. US 150 µl).
Reagent | Sample |
10X Dicer Buffer
|
30 µl
|
RNase-Free Water
|
up to 210 µl
|
Purified dsRNA (60 µg)
|
1-150 µl
|
BLOCK-iT
™ Dicer Enzyme (1 U/µl)
|
60 µl
|
Total volume
|
300 µl
|
Checking the Integrity of d-siRNA
You may verify the integrity of your d-siRNA using polyacrylamide or agarose gel electrophoresis, if desired. We suggest running an aliquot of your dicing reaction (0.5-1 µl of a 300 µl reaction; equivalent to 100-200 ng of dsRNA) on the appropriate gel and comparing it to an aliquot of your starting dsRNA. Be sure to include an appropriate molecular weight standard. We generally use the following gels and molecular weight standard:
What You Should See
When analyzing an aliquot of the dicing reaction by gel electrophoresis, we generally see the following:
Example of Expected Results
In this experiment, purified dsRNA representing a 1 kb region of the lacZ gene was generated following the recommended protocols and using the reagents supplied in the BLOCK-iT™ RNAi TOPO® Transcription Kit. The lacZ dsRNA was diced using the procedure on page 10. Aliquots of the dicing reaction (equivalent to 200 ng of dsRNA) and the initial dsRNA substrate were analyzed on a 4%
E-Gel®.
Results: A prominent band representing d-siRNA of the expected size is clearly visible in the dicing reaction sample (lane 3). This band is not visible in the initial dsRNA substrate sample (lane 2).
Introduction
This section provides guidelines and instructions to purify the d-siRNA produced in the dicing reaction. Use the BLOCK-iT™ RNAi Purification reagents (Box 2) supplied with the kit.
Before proceeding to transfection, note that you must purify the d-siRNA produced in the dicing reaction to remove contaminating long dsRNA duplexes. Transfection of unpurified d-siRNA can trigger the interferon-mediated response and cause host cell shutdown and cellular apoptosis. When purifying d-siRNA, follow the purification procedure provided on page 16 exactly as instructed. This procedure is optimized to allow removal of contaminating long dsRNA and recovery of high yields of d-siRNA.
Experimental Outline
To purify d-siRNA, you will:
The figure below illustrates the d-siRNA purification process.
Advance Preparation
Before using the BLOCK-iT™ RNA Purification reagents for the first time, add 10 ml of 100% ethanol to the entire amount of 5X RNA Wash Buffer to obtain a 1X RNA Wash Buffer (total volume = 12.5 ml). Place a check in the box on the 5X RNA Wash Buffer label to indicate that the ethanol was added. Store the 1X RNA Wash Buffer at room temperature.
The RNA Binding Buffer contains guanidine isothiocyanate. This chemical is harmful if it comes in contact with the skin or is inhaled or swallowed. Always wear a laboratory coat, disposable gloves, and goggles when handling solutions containing this chemical.
Do not add bleach or acidic solutions directly to solutions containing guanidine isothiocyanate or sample preparation waste. Guanidine isothiocyanate forms reactive compounds and toxic gases when mixed with bleach or acids.
Materials Needed
Have the following materials on hand before beginning:
d-siRNA Purification Procedure TOP
Use this procedure to purify d-siRNA produced from dicing 60 µg of dsRNA in a 300 µl reaction volume (see Step 5, Performing the Dicing Reaction). If you have digested < 60 µg of dsRNA and have scaled down the volume of your dicing reaction, scale down the volume of your purification reagents proportionally. For example, if you have digested 30 µg of dsRNA in a 150 µl dicing reaction, scale down the volume of purification reagents used by half.
Important: Before beginning, remove the amount of RNA Binding Buffer needed and add 2-mercaptoethanol to a final concentration of 1% (v/v). Use fresh and discard any unused solution.
Important: When using the d-siRNA, avoid repeated freezing and thawing as d-siRNA can degrade with each freeze/thaw cycle.
Determining the Purity and Concentration of d-siRNA
Use the procedure below to determine the purity and concentration of your purified d-siRNA.
Verifying the Quality of Your d-siRNA
You may verify the quality of your purified d-siRNA using polyacrylamide or agarose gel electrophoresis, if desired. We suggest running a small aliquot of your purified d-siRNA (0.5-1 µl) on the appropriate gel and comparing it to an aliquot of your dicing reaction (equivalent to 100-200 ng of dsRNA). Be sure to include an appropriate molecular weight standard. For recommended gels and a molecular weight standard, we generally use the same gels and molecular weight standard that we use to analyze the quality of the dicing reaction.
If the band representing purified d-siRNA is weak or if you do not see a band, see Troubleshooting for tips to purify your d-siRNA.
Example of Expected Results
In this experiment, the lacZ d-siRNA generated in the dicing reaction depicted in the Example above were purified using the procedure. Aliquots of the purified lacZ d-siRNA (80 ng) and the lacZ dicing reaction (equivalent to 200 ng of dsRNA) were analyzed on a 4% E-Gel®.
Results: A prominent band representing purified d-siRNA of the expected size is clearly visible in lane 3. No contaminating dsRNA or other high molecular weight products remain in the purified d-siRNA sample.
How Much d-siRNA to Expect
The typical yield of d-siRNA obtained from dicing 60 µg of dsRNA (500 bp to 1 kb in size) in a 300 µl dicing reaction ranges from 12-18 µg, with a concentration of 200-300 ng/µl. Note that yields may vary depending on the size and quality of the dsRNA.
Introduction
Once you have purified your d-siRNA, you may perform RNAi analysis by transfecting the d-siRNA into the mammalian cell line of interest, and assaying for inhibition of target gene expression. This section provides general guidelines and protocols to transfect your purified d-siRNA into mammalian cells using the Lipofectamine® 2000 Reagent (Box 3) supplied with the kit. Suggested transfection conditions are provided as a starting point. You will need to optimize transfection conditions to obtain the best results for your target gene and mammalian cell line.
Reminder: You must transfect mammalian cells with purified d-siRNA. Note that transfecting cells with unpurified d-siRNA containing contaminating long dsRNA (i.e. with material directly taken from the dicing reaction) can trigger the interferon-mediated cellular response, resulting in host cell shutdown and cellular apoptosis
.
Factors Affecting Gene Knockdown Levels
A number of factors can influence the degree to which expression of your gene of interest is reduced (i.e. gene knockdown) in an RNAi experiment including:
Take these factors into consideration when designing your transfection and RNAi experiments.
Lipofectamine® 2000 Reagent
The Lipofectamine® 2000 Reagent supplied with the kit is a proprietary, cationic lipid-based formulation suitable for the transfection of nucleic acids including d-siRNA and siRNA into eukaryotic cells (Ciccarone et al., 1999; Gitlin et al., 2002; Yu et al., 2002). Using Lipofectamine® 2000 to transfect d-siRNA into eukaryotic cells offers the following advantages:
Lipofectamine® 2000 is also available separately from Invitrogen
Important Guidelines
Follow these guidelines when transfecting siRNA into mammalian cells using Lipofectamine® 2000:
Materials to Have on Hand
Have the following materials on hand before beginning:
Positive Control
If you are using the BLOCK-iT™ Complete Dicer RNAi Kit, and have diced the control lacZ dsRNA, two options exist to use the resulting purified lacZ d-siRNA for RNAi analysis:
Important: Transfection conditions (i.e. cell density and reagent amounts) vary slightly when d-siRNA and plasmid DNA are co-transfected into mammalian cells.
Transfection Procedure
Use this procedure to transfect mammalian cells using Lipofectamine® 2000. Refer to the table in Recommended Reagent Amounts and Volumes, below for the appropriate reagent amounts and volumes to add for different tissue culture formats. Use the recommended Lipofectamine® 2000 amounts as a starting point for your experiments, and optimize conditions for your cell line and d-siRNA.
- Dilute d-siRNA in the appropriate amount of Opti-MEM® I Reduced Serum Medium without serum. Mix gently.
- Mix Lipofectamine® 2000 gently before use, then dilute the appropriate amount in Opti-MEM® I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature.
- After the 5 minute incubation, combine the diluted d-siRNA with the diluted Lipofectamine® 2000. Mix gently and incubate for 20 minutes at room temperature to allow the d-siRNA:Lipofectamine® 2000 complexes to form (solution may appear cloudy).
Recommended Reagent Amounts and Volumes
The table below lists the recommended reagent amounts and volumes to use to transfect cells in various tissue culture formats. Use the recommended amounts of d-siRNA (see column 4) and Lipofectamine® 2000 (see column 6) as a starting point for your experiments, and optimize conditions for your cell line and target gene.
Note: With automated, high-throughput systems, larger complexing volumes are recommended for transfections in 96-well plates.
Culture Vessel | Relative Surface Area (vs. 24-well) | Volume of Plating Medium | d-siRNA (ng) and Dilution Volume (µl) | d-siRNA Amounts (ng) for Optimization | Lipofectamine®
2000 (µl) and Dilution Volume (µl) | Lipofectamine® 2000 Amounts (µl) for Optimization |
96-well
|
0.2
|
100 µl
|
20 ng in 25 µl
|
5-50 ng
|
0.6 µl in 25 µl
|
0.2-1.0 µl
|
24-well
|
1
|
500 µl
|
50 ng in 50 µl
|
20-200 ng
|
1 µl in 50 µl
|
0.5-1.5 µl
|
6-well
|
5
|
2 ml
|
250 ng in 250 µl
|
100-1000 ng
|
5 µl in 250 µl
|
2.5-6 µl
|
Optimizing Transfection
To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying the cell density (from 30-50% confluence) and the amounts of d-siRNA (see column 5) and Lipofectamine® 2000 (see column 7) as suggested in the tableon the previous page. For cell lines that are particularly sensitive to transfection-mediated cytotoxicity (e.g. HeLa, HT1080), use the lower amounts of Lipofectamine® 2000 suggested in the table above (see column 7).
What You Should See
When performing RNAi experiments using d-siRNA, we generally observe inhibition of the gene of interest within 24 to 96 hours after transfection. The degree of gene knockdown depends on the time of assay, stability of the protein of interest, and on the other factors listed on page 19. Note that 100% gene knockdown is generally not observed, but > 95% is possible with optimized conditions.
For examples of results obtained from RNAi experiments using d-siRNA, see below.
Co-transfecting d-siRNA and Plasmid DNA
If you are using the lacZ d-siRNA as a positive control to assess the RNAi response in your cell line, you will co-transfect the lacZ d-siRNA and the pcDNA™ 1.2/V5-GW/lacZ reporter plasmid into the mammalian cell line and assay for inhibition of b-galactosidase expression after 24 hours. When co-transfecting d-siRNA and plasmid DNA, follow the procedure on the previous page with the following exceptions:
Note: We generally transfect twice the mass of plasmid DNA as d-siRNA.
Culture Vessel | Volume of Plating Medium | d-siRNA (ng) | Plasmid DNA (ng) | Nucleic Acid Dilution Volume | Lipofectamine® 2000 (µl) and Dilution Volume (µl) | Lipofectamine® 2000 Amounts (µl) for Optimization |
96-well
|
100 µl
|
20 ng
|
40 ng
|
25 µl
|
0.6 µl in 25 µl
|
0.2-1.0 µl
|
24-well
|
500 µl
|
50 ng
|
100 ng
|
50 µl
|
2 µl in 50 µl
|
0.5-2.0 µl
|
6-well
|
2 ml
|
250 ng
|
500 ng
|
250 µl
|
10 µl in 250 µl
|
2.5-10 µl
|
Assaying for ß-galactosidase Expression
If you perform RNAi analysis using the control lacZ d-siRNA, you may assay for ß-galactosidase expression and knockdown by Western blot analysis or activity assay using cell-free lysates (Miller, 1972). Invitrogen offers the ß-gal Antiserum (Catalog no. R901-25) and the ß-Gal Assay Kit (Catalog no. K1455-01) for fast and easy detection of ß-galactosidase expression. For an example of results obtained from a ß-galactosidase knockdown experiment, see below.
Note: The ß-galactosidase protein expressed from the pcDNA™1.2/V5-GW/lacZ control plasmid is fused to a V5 epitope and is approximately 119 kDa in size. If you are performing Western blot analysis, you may also use the Anti V5 Antibodies available from Invitrogen (e.g. Anti-V5-HRP Antibody; Catalog no. R961-25 or Anti-V5-AP Antibody, Catalog no. R962-25) for detection
Introduction
Use the information in this section to troubleshoot your dicing, purification, and transfection experiments.
Dicing Reaction
The table below lists some potential problems and possible solutions that may help you troubleshoot the dicing reaction.
Problem | Reason | Solution |
Weak band representing d-siRNA observed on a poly-acrylamide or agarose gel (
i.e. low yield of d-siRNA)
|
Poor quality dsRNA
|
|
Didn’t use enough dsRNA in the dicing reaction
|
| |
dsRNA was degraded
|
| |
Incubated the dicing reaction for longer than 18 hours
|
Do not incubate the dicing reaction for longer than 18 hours.
| |
Incubated the dicing reaction for less than 14 hours
|
Incubate the dicing reaction at 37
°C for 14-18 hours. | |
Smear with molecular weight 21 nt observed on a poly-acrylamide gel
|
Used too much BLOCK-iT
™ Dicer Enzyme in the dicing reaction
|
Follow the recommended procedure to set up the dicing reaction. Do not use more than 60 units of BLOCK-iT
™ Dicer Enzyme in a 300 µl reaction.
|
Incubated the dicing reaction for longer than 18 hours
|
Do not incubate the dicing reaction for longer than 18 hours.
| |
Sample contaminated with RNase
|
| |
No d-siRNA produced
|
dsRNA was degraded
|
|
Sample was contaminated with RNase
|
| |
ssRNA used as substrate
|
If you have used to the BLOCK-iT
™ RNAi TOPO
® Transcription Kit to generate sense and antisense ssRNA, you must anneal the ssRNA to generate dsRNA prior to dicing.
|
Problem | Reason | Solution |
Low yield of purified d-siRNA obtained
|
Eluted d-siRNA from the RNA Spin Cartridge using TE Buffer
|
Elute d-siRNA from the RNA Spin Cartridge using water.
|
Concentration of d-siRNA incorrectly determined
· Sample diluted into water for spectrophotometry
· Sample blanked against water
|
| |
No d-siRNA obtained
|
Forgot to add ethanol to the 5X RNA Wash Buffer
|
Add 10 ml of ethanol to the 5X RNA Wash Buffer (2.5 ml) to obtain a 1X RNA Wash Buffer.
|
Forgot to add isopropanol to the combined flow-throughs from the first RNA Spin Cartridge
|
You must add isopropanol to the combined flow-throughs from the first RNA Spin Cartridge to enable the d-siRNA to bind to the second RNA Spin Cartridge.
| |
Forgot to keep flow-throughs from the first RNA Spin Cartridge
|
Keep the flow-throughs from the first RNA Spin Cartridge. The flow-throughs contain the d-siRNA.
| |
dsRNA present in purified d-siRNA sample
|
Forgot to add isopropanol to the dicing reaction
|
You must add RNA Binding Buffer containing 1% (v/v) b-mercaptoethanol and isopropanol to the dicing reaction to denature the proteins and enable the dsRNA to bind the first RNA Spin Cartridge.
|
Added the mixture containing the flow-through and isopropanol from the first RNA Spin Cartridge back onto the first RNA Spin Cartridge
|
You must add the mixture containing the flow-through and isopropanol from the first RNA Spin Cartridge to a second RNA Spin Cartridge as the first RNA Spin Cartridge contains bound dsRNA.
| |
A260/A280 ratio not in the 1.9-2.2 range
|
Sample was not washed with 1X RNA Wash Buffer
|
Wash the RNA Spin Cartridge containing bound d-siRNA twice with 1X RNA Wash Buffer.
|
RNA Spin Cartridge containing bound d-siRNA not centrifuged to remove residual 1X RNA Wash Buffer
|
Centrifuge RNA Spin Cartridge at 14,000 x g for 1 minute at room temperature to remove residual 1X RNA Wash Buffer and to dry the membrane
. |
Transfection and RNAi Analysis
The table below lists some potential problems and possible solutions that may help you troubleshoot your transfection and knockdown experiment.
Problem | Reason | Solution |
Low levels of gene knockdown observed
|
Low transfection efficiency
|
|
Didn’t wait long enough after transfection before assaying for gene knockdown
|
| |
d-siRNA was degraded
|
| |
Cytotoxic effects observed after transfection
|
Too much Lipofectamine® 2000 Reagent used
|
Optimize the transfection conditions for your cell line by varying the amount of Lipofectamine® 2000 Reagent used.
|
Cells transfected with unpurified d-siRNA
|
Purify d-siRNA using the RNAi Purification reagents supplied with the kit.
Important: Transfecting unpurified d-siRNA is not recommended as the contaminating dsRNA will cause host cell shutdown and apoptosis.
| |
No gene knockdown observed
|
d-siRNA was degraded
|
|
Target region contains no active siRNA
|
Select a larger target region or a different region.
| |
Non-specific off-target gene knockdown observed
|
Target sequence contains strong homology to other genes
|
|