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shRNA and miRNA RNAi vectors

Please review the possibilities below:

  • Single-stranded oligos designed incorrectly; verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top strand oligo.
  • Ensure that oligos are annealed at room temp for 5–10 minutes after heating to 95°C.
  • Check the molar ratio you are using for annealing top and bottom-strand oligo; equal amounts should be used.
  • Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
  • For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences.  The top-strand oligo should include CACC on the 5’ end, while the bottom-strand oligo should include AAAA on the 5’ end.
  • For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5’ end and that the bottom-strand oligo includes CCTG at the 5’ end.

We highly recommend sequencing positive transformants to confirm the sequence of the ds oligo insert. When screening transformants, we find that up to 20% of the clones may contain mutated inserts (generally 1 or 2 bp deletions within the ds oligo). The reason for this is not known, but may be due to triggering of repair mechanisms within E. coli as a result of the inverted repeat sequence within the ds oligo insert. Note: Entry clones containing mutated ds oligo inserts generally elicit a poor RNAi response in mammalian cells. Identify entry clones with the correct ds oligo sequence and use these clones for your RNAi analysis.

Mutated inserts could also be caused by using poor-quality single-stranded oligos. Use mass spectrometry to check for peaks of the wrong mass, or order HPLC- or PAGE-purified oligos to avoid this problem.

Difficulties sequencing could occur because the hairpin sequence is an inverted repeat that can form secondary structure during sequencing, resulting in a drop in the sequencing signal when entering the hairpin. If you encounter difficulties while sequencing, please try the following:

  • Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen™ PureLink® HQ Mini Plasmid Purification Kit (Cat. No. K2100-01) or S.N.A.P.™ Plasmid DNA MidiPrep Kit (Cat. No. K1910-01).
  • Add DMSO to the sequencing reaction to a final concentration of 5%.
  • Increase the amount of template used in the reaction (up to twice the normal concentration).
  • Standard sequencing kits typically use dITP in place of dGTP to reduce G:C compression. Other kits containing dGTP are available for sequencing G-rich and GT-rich templates. If you are using a standard commercial sequencing kit containing dITP, obtain a sequencing kit containing dGTP (e.g., dGTP BigDye® Terminator v3.0 Ready Reaction Cycle Sequencing Kit, Cat. No. 4390229) and use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.

You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.

Low expression levels can be due to several factors.  Please see the suggestions below:

  • Low transfection efficiency: ensure that antibiotics are not added to the media during transfection, and that cells are at the proper cell confluency; optimize transfection conditions by varying the amount of transfection reagent used.
  • Try a time course assay to determine the point at which the highest degree of gene knockdown occurs.
  • Mutations are present in your construct: analyze the transformants by sequencing the ds oligo insert to verify its sequence.
  • Target region is not optimal: select a different target region.
  • Ensure siRNA is designed according to guidelines listed in the respective manual.

Please visit our Transfection Support Center to find technical resources, tips and tricks, and troubleshooting information on transfection.

Inducible RNAi Expression

Please check to ensure that your medium containing fetal bovine serum (FBS) is reduced in tetracycline. Many lots of FBS contain tetracycline, as FBS is often isolated from cows that have been fed a diet containing tetracycline, leading to low basal expression of shRNA. Ensure that a cell line expressing the Tet repressor is being used, and that the cells used are transduced at a suitable MOI. If creating your own Tet repressor–expressing cell line, wait at least 24 hours before transducing cells with your shRNA construct.

Please review the following possibilities:

  • Low transfection efficiency—check plating confluency, amount of plasmid DNA used, and/or reagent used for transfection.
  • Time period—perform a time course of expression to determine the point at which the highest degree of gene knockdown occurs.
  • Ensure that the ds oligo insert is sequence-verified and does not contain mutations.
  • The shRNA sequence is important; you can vary the length of shRNA sequence, change or vary the loop sequence/length, reverse orientation of the shRNA hairpin, or select a different target region. If possible, screen shRNA first by transient transfection first.
  • Make sure that enough tetracycline was added.

If working with the lentiviral construct, please review the additional possibilities below:

  • Polybrene® reagent not included during transduction—ensure that Polybrene® reagent is present when transducing the lentiviral construct into cells.
  • Transduce your lentiviral construct into cells using a higher MOI.
  • Place cells under Zeocin™ selection and generate a stable cell line prior to addition of tetracycline, which can improve gene knockdown results by killing untransduced cells.
  • Titer viral stocks.
  • Store viral stocks correctly at –80°C, do not freeze/thaw more than 3 times, and if stored for longer than 6 months, retiter stock before use. 

Yes, as long as you do not use a cell line expressing the Tet repressor, expressing will be constitutive.

The shRNA chosen may not be working. Verify that the shRNA sequence does not contain > 3 tandem Ts which can cause premature transcription termination. You can try to select a different target region.  Check the hairpin design for the shRNA. Ensure that the correct amounts of tetracycline were added.  Cells should be treated 3–24 hours after transfection with tetracycline to induce shRNA expression.  Assay for the target gene knockdown 24–96 hours following induction. Lastly, please check that the construct was transduced into a T-REx™ repressor-expressing cell line. (To create your own cell line that stably expresses the Tet repressor, use either pcDNA™6/TR (for your pENTR™/H1/TO construct) or pLenti6/TR (for your Lenti4/BLOCK-iT™-DEST construct) and maintain the cell line in medium containing blasticidin.)

Lentiviral Expression

Please see the possible causes and solutions below:

Incorrect antibiotic used to select transformants

Select for transformants on LB agar plates containing 100 µg/mL ampicillin.

Rapid BP/LR reaction may not work for your insert

Use the standard BP and LR reactions.

BP recombination reaction is treated with Proteinase K

Do NOT treat the BP reaction with Proteinase K before the LR reaction.

Did not use the suggested Gateway® BP and LR Clonase® II Plus enzyme mixes, or Gateway® BP and LR Clonase® II Plus enzyme mixes were inactive

Make sure to store the enzymes at –20°C or –80°C. Do not freeze/thaw the enzymes more than 10 times. Use the recommended amounts of enzyme mixes. Test another aliquot of enzyme mix. Test positive control entry clones with DEST vector to confirm whether or not LR Clonase® II is active.

Not enough LR reaction transformed

Transform 2–3 µL of the LR reaction into One Shot® Stbl3™ Chemically Competent E. coli.

Not enough transformation mixture plated

Increase the amount plated.

Did not perform the 1 hour grow out period before plating the transformation mixture

After the heat-shock step, add SOC Medium and incubate the transformation mixture for 1 hr at 37°C with shaking before plating.

Too much BP reaction used in the LR reaction

Use 3 µL BP reaction for the LR reaction.

Did not include a pENTR™ 5’ promoter vector in the LR reaction

You must use either the supplied pENTR™ 5’ promoter vectors (or a pENTR™ 5’ vector you have generated with your own promoter of interest) in the LR reaction.

Some transformants may contain plasmids in which unwanted recombination has occurred between the 5’ and 3’ LTR. We recommend using our One Shot® Stbl3™ Chemically Competent E. coli cells, as they help in stabilizing lentiviral DNA containing direct repeats, and generally give rise to fewer unwanted recombinants. We recommend screening both colony sizes; however, in general, for lentiviral plasmids the small colonies tend to be the correct clones. Large colonies may have undergone a recombination event to delete part of the plasmid, thus allowing the cells to grow faster.

Ensure that the competent cells used were stored properly at –80°C, and thawed on ice for immediate use. When adding DNA, mix competent cells gently: do not mix by pipetting up and down. Also do not exceed the maximum recommended amount of DNA for transformation (100 ng) or allow the volume of DNA added to exceed 10% of the volume of the competent cells, as these may inhibit the transformation. Visit our Transfection Support Center to for more troubleshooting tips.

Please review the following possibilities and solutions:

Reason

Solution

Low transfection efficiency

  • Used poor-quality expression construct plasmid DNA
  • Unhealthy 293FT cells used
  • Cells transfected in medium containing antibiotics (i.e., Geneticin®)
  • Incorrect ratio of plasmid DNA to transfection reagent
  • 293FT cells plated too sparsely

 

  • Do not use plasmid DNA from a miniprep for transfection.  We recommend using the PureLink® HiPure Plasmid DNA Purification Midiprep Kit to prepare plasmid DNA
  • Use healthy 293FT cells under passage 20; do not overgrow
  • Use a DNA (in µg): Lipofectamine® 2000 (in µL) ratio ranging from 1:2 to 1:3
  • Plate cells such that they are 90–95% confluent at the time of transfection, or use the recommended transfection protocol (i.e., add cells to medium containing DNA:lipid complexes)

Transfected cells not cultured in medium containing sodium pyruvate

One day after transfection, remove medium containing DNA:lipid complexes and replace with complete medium containing sodium pyruvate. Sodium pyruvate provides an extra energy source for the cells.

Lipofectamine® 2000 Reagent handled incorrectly

Store at 4°C; do not freeze. Mix gently by inversion before use. Do not vortex.

Viral supernatant harvested too early

Viral supernatants can generally be collected 48–72 hours posttransfection. If many cells are still attached to the plate and look healthy at this point, wait an additional 24 hours before harvesting the viral supernatant.

Viral supernatant too dilute

Concentrate virus using any method of choice (Yee, 1999).

Viral supernatant frozen and thawed multiple times

Do not freeze/thaw viral supernatant more than 3 times.

Poor choice of titering cell line

We recommend using HT1080 cells.

Target gene is essential for cell viability

Make sure that your target gene is not essential for cell viability or growth by performing a transient transfection with the entry construct containing the miRNA of interest.

Polybrene® reagent not included during titering procedure

Transduce the lentiviral construct into cells in the presence of Polybrene® reagent.

Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at –80°C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral construct into cells in the presence of Polybrene® reagent.

Please see the possible reasons and solutions below:

Reason

Solution

Low transduction efficiency

  • Polybrene® reagent not included during transduction
  • Nondividing cell type used

 

  • Transduce the lentiviral construct into the cells in the presence of Polybrene® reagent.
  • Transduce your lentiviral construct into cells using a higher MOI.

MOI too low

Transduce your lentiviral construct into cells using a higher MOI.

Cells harvested and assayed too soon after transduction.

Do not harvest cells until at least 48–72 hours after transduction to allow expressed miRNA to accumulate in trasduced cells. If low levels of knockdown are observed at 48 hours, culture cells for a longer period of time before assaying for gene knockdown or place cells under blasticidin selection. Note: Placing cells under blasticidin selection can improve gene knockdown results by killing untransduced cells.

Target gene is important for cell viability

Make sure that your target gene is not essential for cell viability or growth.

Viral stocks not titered

Titer the lentivirus.

Viral stock stored incorrectly

Aliquot and store at –80°C; Do not freeze/thaw more than 3 times; if stored for longer than 6 months, retiter stock before use.

miR RNAi with weak activity chosen

Select a different target region. Perform a transient transfection with the miR RNAi expression construct to verify its activity.  After doing so, proceed to generate lentivirus.  Please note, in general, transient transfection greatly overexpresses miR RNAi sequences, so moderately active expression clones may be less active when expressed from a lentiviral construct.

Viral stocks stored incorrectly

Aliquot and store at –80°C. Do not freeze/thaw more than 3 times.

MOI too low

Transduce your lentiviral construct into cells using a higher MOI.

Cytotoxic effects can result due to several possibilities.  Please see the possible reasons and solutions below:

Reasons

Solutions

Target gene is essential for cell viability

Make sure that your target gene is not essential for cell viability or growth.

Large volume of viral supernatant used for transduction

Remove the “spent” medium containing virus and replace with fresh, complete medium; concentrate the virus.

Polybrene® reagent used during transduction

Verify the sensitivity of the cells to Polybrene® reagent; if cells are sensitive, omit the Polybrene®.

Too much blasticidin used for selection

Determine the blasticidin sensitivity of your cell line by performing a kill curve experiment. Use the minimum blasticidin concentration required to kill your untransduced cell line.

The target sequence used may contain strong homology to other genes; please select a different target region.

Please ensure that the recommended filter sets for detection of fluorescence are used. Use an inverted fluorescence microscope for analysis. If desired, allow the protein expression to continue for 1–3 days before assaying for fluorescence.

Adenoviral Vector Expression

Here are possible causes and solutions:

Cause

Solution

Incorrect antibiotic used to select for transformants

Select for transformants on LB agar plates containing 100 μg/mL ampicillin.

LR recombination reaction not treated with proteinase K

Treat reaction with proteinase K before transformation.

pAd/BLOCK-iT™-DEST plasmid DNA was sheared

Use care when handling the adenoviral Destination vector. Do not perform

excessive manipulations (e.g., vortexing or pipetting the solution vigorously) that may shear the DNA.

Didn’t use the suggested amount of LR Clonase® II enzyme mix, or LR Clonase® II enzyme mix was inactive

-Make sure to store the LR Clonase® II enzyme mix at –20°C.

-Do not freeze/thaw the LR Clonase® II enzyme mix more than 10 times.

-Use the recommended amount of LR

Clonase® II enzyme mix (see page 14 of the manual).

-Test another aliquot of the LR Clonase® II enzyme mix.

Not enough LR reaction transformed

Transform 2–3 μL of the LR reaction into the appropriate competent E. coli strain. Use

E. coli cells with a transformation efficiency >1 x 108 cfu/μg.

Not enough transformation mixture plated

Increase the amount of E. coli plated.

Too much entry clone DNA used in the LR reaction

Use 50–150 ng of the entry clone in the LR reaction.

Here are possible causes and solutions:

Cause

Solution

LR reaction transformed into an E. coli strain containing the F’ episome and the ccdA gene

Use an E. coli strain that does not contain the

F’ episome for transformation (e.g., TOP10,

DH5α-T1R).

Deletions (full or partial) of the ccdB gene from the pAd/BLOCK-iT™-DEST vector

The adenoviral DEST vectors are provided in solution and are ready-to-use in an LR reaction. However, if you wish to

propagate them:

-Select for transformants in medium containing 100 μg/mL ampicillin and 15–30 μg/mL chloramphenicol, to maintain the integrity of the vector.

-Prepare plasmid DNA from one or more colonies and verify the integrity of the vector before use.

Here are possible causes and solutions:

Cause

Solution

Low transfection efficiency:

-pAd/BLOCK-iT™-DEST expression clone plasmid DNA was sheared

 

 

-Incomplete PacI digestion or digested DNA contaminated with phenol, ethanol, or salts

-Unhealthy 293A cells; cells exhibit low viability

-293A cells plated too sparsely on the day before transfection

-Incorrect ratio of plasmid DNA to transfection reagent ratio

 

-Use care when handling plasmid DNA. Do not perform excessive manipulations (e.g., vortexing or pipetting the solution vigorously) that may shear the DNA.

-Repeat the PacI digestion. Make sure purified DNA is not contaminated with phenol, ethanol, or salts.

-Use healthy 293A cells; do not overgrow cells.

-Cells should be 90–95% confluent at the time of transfection.

-Optimize such that plasmid DNA (in μg):Lipofectamine® 2000 (in μL) ratio ranges from 1:2 to 1:3. If you are using another transfection reagent, optimize according to the manufacturer’s recommendations.

Viral supernatant too dilute

Concentrate virus using CsCl purification or any method of choice.

Viral supernatant frozen and thawed multiple times

Do not freeze/thaw viral supernatant more than 10 times.

Here are possible causes and solutions:

Cause

Solution

Viral stocks stored incorrectly

Aliquot and store stocks at –80°C. Do not freeze/thaw more than 10 times.

Incorrect titering cell line used

Use the 293A cell line or any cell line with the characteristics discussed on page 23 of the manual.

Agarose overlay incorrectly prepared

Make sure that the agarose is not too hot before addition to the cells; hot agarose will kill the cells.

This could be due to insufficient dilution of the viral supernatant. We recommend titering the adenovirus stock using 10-fold serial dilutions ranging from 10–4 to 10–9.

Here are possible causes and solutions:

Cause

Solution

Viral stocks stored incorrectly

Aliquot and store stocks at –80°C. Do not freeze/thaw more than 10 times.

Gene of interest contains a PacI site

Perform mutagenesis to change or remove the PacI site.

shRNA with no activity chosen

Select a different target region. If possible, screen shRNA first by transient transfection.

MOI too low

Transduce your adenoviral construct into cells using a higher MOI.

Here are possible causes and solutions:

Cause

Solution

Poor transduction efficiency:

-Mammalian cells not healthy

-Nondividing cell type used

 

-Make sure that your cells are healthy before transduction.

-Transduce your adenoviral construct into cells using a higher MOI.

MOI too low

Transduce your adenoviral construct into cells using a higher MOI.

Low viral titer

Amplify the adenoviral stock using the procedure on page 27 of the manual.

shRNA with weak activity chosen

Select a different target region. If possible, screen shRNA first by transient transfection.

Adenoviral stock contaminated with RCA (replication-competent adenovirus)

-Screen for RCA contamination.

-Prepare a new adenoviral stock or plaque-purify to isolate recombinant adenovirus.

Cells harvested too soon after transduction

Do not harvest cells until at least 48–72 hours after transduction to allow expressed shRNA to accumulate in transduced cells.

Cells harvested too long after transduction

For actively dividing cells, assay for maximal levels of recombinant protein expression within 5 days of transduction.

Knockdown of gene of interest causes cell death

Make sure that your target gene is not essential for cell viability or growth.

Here are possible causes and solutions:

Cause

Solution

Too much crude viral stock used

-Reduce the amount crude viral stock used for transduction or dilute the crude viral stock.

-Amplify the adenoviral stock.

-Concentrate the crude viral stock.

Wild-type RCA (replication-competent adenovirus) contamination

Screen for RCA contamination. Plaque-purify to isolate recombinant adenovirus, or prepare a new adenoviral stock.