Cdnadirect Kit Manual: Cat No.: 0667800021730

cDNAdirect Kit GeNei™ cDNAdirect Kit GeNei™
cDNAdirect Kit
Manual
Cat No.: 0667800021730

Revision No.: 01070219
Genei Laboratories Private Limited, 2019 Genei Laboratories Private Limited, 2019
Notes:
CONTENTS
 Description 1
 Materials Supplied 4
 Protocol 7
 Troubleshooting 14
 Ordering Information 17
 Flow Chart 18
Description: Flow Chart:

cDNAdirect Kit is an optimized procedure to synthesize first-
strand cDNA directly from cultured mammalian cells without
isolating RNA. The synthesized first-strand cDNA is ready to
be used in PCR, RT-PCR and other downstream applications
Harvest cell
such as quantifying mRNA level from a small number of cells.
In cDNAdirect Kit, cultured cells are washed in PBS to
remove cell culture medium and extracellular contaminants.
Cells are then incubated with specially designed Lysis Buffer
at 75°C to rupture the cells and inactivate the endogenous
RNases. The crude cell lysate is treated with DNase I to
degrade the genomic DNA and is ready to use for first strand Lyse at 750C for 14 minutes and treat
cDNA synthesis and PCR using either a one-step or two-step with DNase I. Inactivate DNase I at 75°C
Reverse Transcriptase-PCR.

Highlights:
 No RNA isolation or purification required.
 Simple protocol and generates high quality cDNA for use Add RH6 or Oligo (dT)18 and other RT
in a variety of downstream applications. reagents
 Kit is optimized for synthesizing cDNA from small number
…...TTTT oligo (dT) 18
of cells. …..AAAA 3’ mRNA
5’
 Synthesized cDNA is free of genomic DNA contamination.
 Single step procedure to recover cDNA in 30 minutes. First strand cDNA synthesis
 Allows detection of rare transcripts. ….AAAA
 Compatible with wide range of mammalian cell types. ..….TTTT
Time Required: Denature at 95°C for 5-10 minutes

Preparation Time : 30 minutes
Hands on Time : 60 minutes
Single stranded cDNA
1 RT-PCR 18
Ordering Information: M 1 2 3 4 5 6 7 8 9 10 11 12 M
Lane M :100 bp DNA ladder ,

Product Size Cat No. Lanes 1,3,5: gapdh amplification from HeLa ,
MCF 7 and NIH 3T3 respectively ,
Lanes 2,4,6: β-actin amplification from HeLa ,
cDNAdirect Kit 1 EA 0667800021730 MCF 7 and NIH 3T3 respectively .
Lanes 7-12: Negative RT-PCR.
(20 Preparations)
Fig. 1. RT-PCR amplification of GAPDH and β-actin genes in different

Other Related Products: cell lines using cDNA direct technique.
Cat No. Related Products
2117100021730 GeneiPure Total RNA Isolation Kit- Cells
M 1 2 3 4 5 6 7 Lane M : 100 bp DNA ladder
and Tissues
Lane 1 : 326 bp fragment of RXRB gene
2151100011730 Water (Protease, DNase, RNase free) Lane 2 : 305 bp fragment of CASP9 gene
0655600011730 DNase I (RNase free) Lane 3 : 304 bp fragment of p53 gene
1101200011730 Recombinant RNase inhibitor Lane 4 : 309 bp fragment of c-myc gene
0602300031730 Taq DNA Polymerase (5U/ µl) Lane 5 : 208 bp fragment of PCNA gene
Lane 6 : 223 bp fragment of GAPDH gene
0600900021730 AMV Reverse Transcriptase Lane 7 : 405 bp fragment of β–actin gene
0601300051730 M-MuLV Reverse Transcriptase
0602200031730 PCR Master Mix (2X) Fig. 2. RT-PCR amplified products of different genes in
0693100011730 RT-PCR Primer Sets NIH-3T3 cell line using cDNAdirect Kit.
Lane M : 100 bp DNA ladder

M 1 2 3 4 5 6 7 8 9 M Lane 1 : Amplifcation from 800 HeLa cells
Email:
Lane 2 : Amplifcation from 400 HeLa cells
Sales: sales@geneilabs.com Lane 3 : Amplifcation from 200 HeLa cells
Lane 4 : Negative RT-PCR HeLa
Customer Support: techsupport @ geneilabs.com Lane 5 : Positive RT-PCR HeLa
Lane 6 : Amplifcation from 800 MCF-7cells
Lane 9 : Negative RT-PCR MCF-7
Fig. 3. RT-PCR amplification of -actin gene fragment (323 bp) from

different number of HeLa & MCF-7 cells using cDNAdirect Kit.
17 2
M 1 2 3 4
Possible Cause Comments
Check if PCR for the gene of interest
works with your PCR primers,
reagents, and equipment by using
Lane M : 100 bp DNA ladder purified RNA from the same (or
Lane 1 : 598 bp amplification of oct-4
Lane 2 : 468 bp amplification of nanog
similar) source in PCR. If there is no
Sample does not good amplification obtained using
Lanes 3 & 4 : Negative RT-PCR.
contain mRNA cDNA from purified RNA, it will not
work with cDNA from cDNAdirect Kit.
Verify the protocol for cDNAdirect Kit
Fig. 4A. Gene expression in Mouse Embryonic Stem Cells. Kit by including an aliquot of Positive
Amplification of mouse transcription factor coding gene fragments
from mouse embryonic stem cell line(R1GC2).
RNA control.
Unexpected PCR products in RT-PCR
M 1 2 3 4 5 6 may arise from alternate splice
variants of a transcript or amplification
Lane M : 100 bp DNA ladder
Lane 1 : β-actin gene fragment
Unexpected of a fragment from related message.
from 10 ES cells; targets in the Cloning and sequencing of the PCR
Lane 2 : β-actin gene fragment cDNA products would solve this problem.
from 100 ES cells; Alternatively, design primers to a
Lane 3 : β-actin gene fragment
from 500 ES cells unique region of the transcript (e.g.
Lanes 4-6 : Negative RT-PCR. the 3’ UTR), and try these in PCR.
Reverse Check reaction components by doing
Fig. 4B. Amplification of mouse β-actin gene from low number of Transcription a positive control reaction.
R1GC2 mouse embryonic stem cell line reaction or PCR
components are
not working
Observation: RT-PCR product in Negative control(s)

Possible cause Comments
Ineffective DNase I treatment DNase treatment can be
made more rigorous by
increasing the incubation
time to 30 minutes at step
19.
DNA contamination in PCR Check reagents by setting
reagents up a negative PCR.
3 16
Possible Cause Comments Materials Supplied:

Volume of Amplification efficiency of PCR The list below provides information about the materials
Reverse decreases as amount of Reverse supplied in the kit. The product should be stored as
Transcription Transcription product increases. suggested.
reaction added Generally the volume of Reverse
to PCR too high Transcription product added should Quantity
not exceed 10% of the final PCR Materials Store
volume. 0667800021730
This reaction should be carried out at (20 Preps.)
42°C. When analyzing RNAs with a 10X PBS 4 ml -20°C
Temperature of high degree of secondary structure, it cDNAdirect Kit Lysis Buffer 2 ml -20°C
Reverse may be advantageous to increase the DNase I (RNase free) 80 µl -20°C
Transcription temperature to 50°C. However, 
reaction temperatures exceeding 42°C will cDNAdirect Kit RT mix 160 µl -20°C
reduce the activity of AMV RT and AMV Reverse Transcriptase 20 µl -20°C
may therefore affect the cDNA yield. RNase inhibitor 20 µl -20°C
Pipetting error or Check the pipettes before setting up Random Hexamer 40 µl -20°C
reagent missing the experiment. Mix all reagents
when setting up properly after thawing and repeat the Positive RNA control 5 µl -20°C
Reverse Reverse Transcription reaction. Positive control sense primer 0.5 µg -20°C
Transcription Positive control antisense 0.5 µg -20°C
reaction primer
Short incubation A minimum of 30-minute incubation Nuclease Free Water 2x1 ml -20°C
time in Reverse time is required.
Transcription
reaction Materials required but not supplied:
Equipments : Refrigerated micro centrifuge for 1.5 ml tubes,
Inappropriate If using gene specific primers, check
Micro centrifuge for PCR tubes, Incubator,
concentration or the concentration and integrity of the
Thermal cycler, Haemocytometer
degradation of primer. If required, perform Reverse
Reagents : Mammalian cell cultures,
primers of Transcription reactions with different
Trypsin (for adherent cell cultures only),
Reverse primer concentrations.
Cell culture media, Taq DNA Polymerase,
Transcription
dNTPs, Taq buffer, Gene specific primers,
reaction
DEPC treated water,
RNase was not It is important that the sample in
10X PBS (for cell culture)
completely cDNAdirect Kit Lysis Buffer reach Other Requirements : RNase free vials and tips,
inactivated in the 75°C quickly to effectively inactivate 1.5 ml vials, Pipettes,
sample endogenous RNases. Ice, Gloves
15 4
Notes: Troubleshooting:
 Read the entire procedure carefully prior to
starting the experiment.
Observation: No product or unexpected products detected in
 Wear gloves at all times during the experiment.
RT-PCR
Change gloves frequently.
Possible Cause Comments
 Cell Types: This kit has been tested for different
Inappropriate Seed multiwell plate with different
mammalian cell lines, including
number of cells number of cells. Determine which cell
 HeLa (Human) seeded number gives optimum PCR results.
 COS-7 (African Green Monkey) Cells not washed Wash cells to remove extracellular and
 MCF-7 (Human) with 1X PBS intracellular contaminants. Ensure that
 NIH-3T3 (Mouse) the PBS used is ice cold.
 Cell concentration: The maximum cell concentration Inappropriate Appropriate volume of lysis buffers
varies according to the type of cell. To obtain volume of should be added for complete lysis.
maximum sensitivity, the optimal cell concentration cDNAdirect (Refer Table 1 at step 13)
should be <1000-cells/µl of cDNAdirect Kit Lysis Lysis Buffer
Buffer. At higher concentration it may be possible to added to cells
amplify the message but a less intense band may be Reverse Carry out all Reverse Transcription
produced compared to the optimal cell number. Transcription reactions on ice.
Determining the ideal cell concentration is crucial. If reaction setup in
the cell concentration is too high, RNases in the an inappropriate
sample may not be totally inactivated and/or high manner
concentration of cellular components (debris) may PCR cycles are too few.
inhibit Reverse Transcription and can give less intense The sensitivity of PCR increases by
band or no amplification. increasing the number of PCR cycles.
 Cell lysis: cDNAdirect Kit Lysis Buffer has been Primer annealing temperature is not
specially formulated to facilitate cell lysis. It is optimal.
important that Lysis Buffer should be ice cold or An unexpected product in RT-PCR
thawed immediately prior to use to protect the RNA PCR is not
comes from non-specific priming of
from degradation because RNases have minimal optimized
unrelated cDNA sequences during
activity at 0°C. It is recommended to lyse the cells in PCR. Raising the stringency by
100 µl volume of Lysis Buffer so that the temperature increasing the annealing temperature
of the cell lysate will rise to 75°C rapidly. Heating step may improve results. Try different
should be performed in thermal cycler preheated to annealing temperatures to identify the
75°C in a 0.5 ml PCR tube for complete RNase one that works the best.
inactivation.
5 14
PCR conditions for samples:  Handling RNA: RNases are very stable and active
enzymes that generally do not require cofactors to
Annealing
94°C 94°C
temperature*
72°C 72°C function. It is very difficult to inactivate RNases and
even minute amounts are sufficient to degrade RNA.
30 seconds- 30 seconds- 7.0 -10
2.0 minutes 30 seconds
1minute 1 minute minutes
The plastic ware and glassware in use must be RNase
free. Extensive care should be taken to avoid
Initial Final
35-40 cycles
Extension
introduction of RNases into the RNA sample during
Denaturation
the procedure. In order to create and maintain an
* Annealing temperature would vary with the type of primer RNase free environment, the following precautions
used. must be taken.
a. Proper aseptic technique should always be used
Note: when working with RNA.
 Annealing temperature of control reaction is b. Hands and dust particles which carry bacteria and
60°C. molds are most common sources of RNase
 Product size with control primers is 472 bp. contamination. It is always recommended to wear
latex or vinyl gloves while handling RNA samples
36. Maintain the reaction at 4°C after cycling. The to prevent RNase contamination from skin or from
sample can be stored at -20°C until use. dusty laboratory equipments. The gloves should
be changed frequently and the tubes should be
kept closed whenever possible.
Appendix: c. The use of sterile, disposable polypropylene tubes
is recommended throughout the procedure. These
DEPC treated water tubes are RNase free and do not require
pre-treatment to inactivate RNases.
Add 1 ml of DEPC solution to 1 litre of sterile distilled water.  DNase I treatment and inactivation: DNase
Mix thoroughly on a magnetic stirrer overnight. Autoclave two treatment is very important to remove genomic DNA
times to remove traces of DEPC. from the cell lysate. Otherwise, PCR products
generated from genomic DNA will be indistinguishable
from those amplified from cDNA and minus-RT control
will produce PCR product, especially if there are
pseudogenes for the target gene in the genome. It is
important to completely inactivate DNase I by heating
to 75°C for 5 minutes. Partial inactivation would lead
to degradation of products generated by Reverse
Transcription.
 Positive and negative controls for cDNA synthesis and
PCR amplification should be prepared as given in the
protocol.
13 6
 Dilute 10X PBS (provided) to 1X PBS with DEPC 30. Assemble the following components in a sterile 0.2
treated water. i.e., to 1 ml of 10X PBS add 9 ml of or 0.5-ml PCR tube or plate well at room temperature
DEPC treated water to get 1X PBS. Store the 1X or on ice. For multiple reactions, prepare a master
PBS at 4°C. (Refer to the appendix for preparation of mix of common components.
DEPC water). The PBS provided is used to remove Note: PCR components are not supplied.
cell culture medium and other extracellular
contaminants. Make a cocktail of the PCR components as indicated below,
 The thermal cycler should be kept ready at 75°C at aliquot them
the start of the experiment.
 Oligo (dT) 18 primers can also be used instead of Components Volume Final Conc.
Random Hexamer for Reverse Transcription. 10X PCR buffer 2.5 µl 1X
 Positive RNA control is provided for checking cDNA 10mM dNTP mix 0.5 µl 0.2 mM
synthesis and PCR reaction. Sense primer 1 µl 100 ng
 PBS for use in cell culture is not provided with the kit. Anti-sense primer 1 µl 100 ng
 PCR components are not provided in the kit. Taq DNA Polymerase (1U/µl) 1 µl 1.0 U
Protocol: Note: For minus-template PCR control reaction

Cell Harvesting use Nuclease Free Water instead of cDNA from
The following procedure is suitable for lysing adherent cell Reverse Transcription reaction.
cultures in vessels larger than 24-well plate wells. For cells in
suspension, skip Steps 1–4 and proceed to Step 5 below. 31. Add 5-10 µl of cDNA to the respective tubes. Make
 For Adherent Cell Cultures: up the final volume to 25 µl with Nuclease Free
1. Add enough trypsin to cover the adherent cells in the Water.
tissue culture dish, plate, or flasks (e.g., for a 10-cm 32. For PCR with Positive control RNA use 1 µl each of
dish, use ~1 ml; for a T75 flask, use ~3 ml). Positive control sense primer and antisense primer.
Note: Excess use of trypsin can be toxic for the 33. Mix contents of the tube and centrifuge briefly to
cells. collect the contents.
2. Incubate for 5 minutes in a 37°C incubator. 34. Start preheating the thermal cycler to 95°C, and put
3. Check for cell detachment under a microscope. If the tubes in the thermal cycler when the temperature
cells have not detached, gently tap the dish or flask has reached >85°C.
to dislodge the cells, or let the cells incubate longer, 35. Perform 35-40 cycles of PCR amplification as
checking them every minute under a microscope. indicated:
4. When all the cells have detached, add 2X volume of
serum-containing media. Swirl gently to inactivate
the trypsin.
Note: The media must contain serum to inactivate
the trypsin.
12
7
26. Add the following Reverse Transcription reagents: 5. Pipette the cells gently up and down to mix, and then
8 µl cDNAdirect Kit RT mix transfer the cell suspension to a 15 ml polypropylene
1 µl RNase inhibitor tube.
1 µl AMV RT enzyme 6. Spin the tube at 1,500 rpm for 5 minutes to pellet the
Mix gently and centrifuge briefly. cells.
Note: Set up a negative Reverse Transcriptase 7. Aspirate the media and wash the cell pellet with
control with 1 µl of sterile distilled water instead 5-10 ml of 1X PBS. (Not provided with the kit).
of AMV-RT enzyme. 8. Spin the tube at 1,500 rpm for 5 minutes to pellet the
27. Incubate at 42°C for 60 minutes. cells.
28. Incubate at 95°C for 5-10 minutes to inactivate the 9. Aspirate the PBS and resuspend the pellet in 1 ml of
Reverse Transcriptase enzyme. culture media. Mix the cell suspension gently.
29. Store reaction at -20°C or proceed directly to PCR Note: Inappropriate mixing of cells may lead to
amplification step. cell clumping and incomplete cell lysis.
Note: cDNA can be stored at -20°C for a month. It 10. Collect a small aliquot to verify that the cells are at
is recommended to store cDNA as aliquots to the desired concentration. Determine cell density
avoid repeated freeze thaws of the cDNA. manually using a Hemocytometer chamber.
11. Adjust the cell density using ice-cold 1X PBS
PCR Amplification: (provided as 10X stock) so that it falls within the
Set up the following PCR-control reactions to check different range of 1–5,000 cells/µl.
parameters of experiment: 12. Transfer 2-10 µl of cells (<50,000 cells) to the PCR
a. Positive control: To check all the reagents and tube/well.
procedure, take cDNA from Positive RNA control
(provided) as a PCR template for PCR reaction.  For Cells in suspension:
b. Negative control: Include two negative controls: Skip steps 1-4. Follow the protocol from step 5-12.
 Negative Reverse Transcriptase control to check the Note: It is recommended to start the experiment with
genomic DNA contamination in cell lysate and freshly harvested cells.
Reverse Transcription reagents. (Use template from
step 26).
Cell Lysis
 Minus template PCR Control to verify that none of
13. Add 100 µl ice-cold cDNAdirect Kit Lysis Buffer to
the PCR reagents are contaminated with gDNA. Use
Nuclease Free Water instead of cDNA from Reverse the cells, taken in PCR tubes on ice and mix by
Transcription reaction. gentle vortexing or pipetting. Keep the tubes on ice
until cDNAdirect Kit Lysis Buffer has been added to
all the samples.
Note: It is recommended to reduce the volume
of Lysis Buffer with the starting material
(Refer Table 1).
8
11
Table 1: 18. Mix the solution thoroughly by gently pipetting up

No. of cells (starting material) Volume of cDNAdirect and down. Centrifuge briefly to bring the solution to
Kit Lysis Buffer (µl) the bottom of the tube or the plate.
10 cells or more 10 19. Incubate the DNase I reaction at 37°C for 15 minutes
100-500 50 to remove any genomic DNA contamination in the
3 4
10 -10 100 cell lysate.
Note: A longer incubation time (up to 30 minutes)
14. Immediately transfer the tube/plate to an incubator or
may be used for larger samples (>5,000 cells).
thermal cycler preheated to 75°C and incubate for
However, longer incubation times may greatly
14 minutes.
Note: Do not exceed the incubation time to more reduce cDNA yield.
than 14 minutes as excess incubation with Lysis 20. Inactivate the DNase I by heating at 75°C for 5
Buffer will affect the cDNA synthesis. minutes.
Note: It is important to completely inactivate
15. After incubation, immediately place tubes on ice for
DNase I by heating to 75°C for 5 minutes. Partial
~1 minute.
inactivation would lead to degradation of
16. Spin briefly to collect the condensation and proceed
products generated by Reverse Transcription.
to DNase I treatment.
21. Centrifuge briefly and use this cell lysate directly for
Positive Control cDNA synthesis.
Note: Cell lysate can be stored at - 70°C for few
For the positive control reaction, add 1 µl of the positive RNA
weeks as aliquots. Do not freeze thaw the cell
control provided to the PCR tube or plate well instead of cell
lysate more then once.
lysate.
cDNA Synthesis
DNase I treatment 22. Place each tube from DNase I treatment on ice, and
17. Add 4 µl DNase I (1U/µl) per 100 µl of cDNAdirect use 5-8 µl cell lysate with 2 µl of the supplied
Kit Lysis Buffer. The final DNase I concentration Random Hexamer. Make up the final volume to 10 µl
should be 0.04U/µl of cDNAdirect Kit Lysis Buffer. with Nuclease Free Water in a PCR tube.
Note: In case of samples where Lysis Buffer is Note: Oligo (dT) 18 primers can also be used
<100 µl add DNase I such that final concentration instead of Random Hexamer for Reverse
is 0.04 U/µl of cDNAdirect Lysis Buffer Transcription. If you use a gene specific primer,
(Refer Table 2). its final concentration should be 0.25-5 µM.
Table 2 : 23. Incubate the tube at 70°C for 4 minutes in a thermal
No. of Cells Volume of cDNAdirect DNase I cycler. Immediately place the tube on ice for
(starting material) Kit Lysis Buffer (µl) (µl) 2 minutes.
24. Centrifuge the tube briefly to collect the
10 cells or more 10 0.4
condensation.
100-500 50 2
3 4 25. Thaw cDNAdirect Kit RT mix on ice. Mix thoroughly
10 -10 100 4
before use.
9
10

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Cdnadirect Kit Manual: Cat No.: 0667800021730

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cDNAdirect Kit GeNei™ cDNAdirect Kit GeNei™

Cat No.: 0667800021730

Description: Flow Chart:

Time Required: Denature at 95°C for 5-10 minutes

Single stranded cDNA

Lane M :100 bp DNA ladder ,

Fig. 1. RT-PCR amplification of GAPDH and β-actin genes in different

Lane M : 100 bp DNA ladder

Fig. 3. RT-PCR amplification of -actin gene fragment (323 bp) from

Observation: RT-PCR product in Negative control(s)

Possible Cause Comments Materials Supplied:

Protocol: Note: For minus-template PCR control reaction

Table 1: 18. Mix the solution thoroughly by gently pipetting up

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