Product FAQ

I’m setting up my RT reaction and am trying to decide whether I should use random primers, oligo(dT) primer, gene-specific primer, or oligo(dT)/random mix primers. What would you suggest?

Answer

Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.

Answer Id: E7313

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Product FAQ

How does the Anchored Oligo(dT)20 Primer differ from standard oligo(dT) primers?

Answer

Anchored Oligo(dT) primers have 2 random bases at the 3' end of the stretch of Ts. The first random base is either an A, G, or C, while the second can be any of the 4 standard nucleotides, i.e., A, T, G or C. The use of anchored oligo(dT) primers results in increased cDNA synthesis yields.

Answer Id: E4223

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SDS

BP OLIGO DT PRIMER

Catalog #
  • 18249029(Discontinued)
  • 450002(Discontinued)
  • 56441
  • A1118001

Manual / Product Insert

User Guide: Anchored Oligo dT Primers

Version: Jan. 2015
Catalog #
  • AB1247(Discontinued)

Product FAQ

What is the highest temperature that SuperScript III , SuperScript II, MMLV, or ThermoScript can be used?

Answer

The optimal temperature for SuperScript III RT is 50 degrees C, and can be used up to 55 degrees C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. Optimal temperature for MMLV is 42 degrees C. ThermoScript RT shows optimal activity at 60 degrees C, and can be used at temperatures as high as 70 degrees C (for amplicons expected to be 1 kb or less). For PCR products expected to be greater than 1 kb, a maximum first strand synthesis temperature of 60-65 degrees C is suggested. Be sure your first-strand primer anneals at the high temperature, especially when gene-specific primers are used for high-temperature stable reverse transcriptases. We recommend oligo (dT)20 for cDNA synthesis when using an oligo (dT) primer for first-strand synthesis with these enzymes.

Answer Id: E2928

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Product FAQ

How can reverse transcriptases be inactivated?

Answer

The enzymes can be inactivated by adding a chelating agent such as EDTA. Alternatively, with the exception of ThermoScript RT and Thermo-X RT, the enzymes can be heat inactivated at 70 degrees C for 10 min.

ThermoScript RT should be heated to 85 degrees C for 5 min for complete inactivation.

For Thermo-X RT, if using an oligo(dT) primer, add EDTA to the reaction at a final concentration of 5 mM. Inactivate the reaction by heating at 90 degrees C for 5 min.

Answer Id: E2957

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Product FAQ

I’m getting no RACE PCR products. What could be the cause of this and what do you suggest I try to fix this?

Answer

Here are some recommendations:

-Check the HeLa control RNA; RNA degradation or failure of either the PCR or RT reaction may produce no RACE PCR products.
-Your gene may be in low abundance: increase the number of PCR cycles or perform nested PCR.
-Your gene is not expressed in this tissue: amplify with two GSPs to assay for the presence of your gene’s cDNA.
-RT reaction failed to generate cDNA for your gene: perform RT with random primers or a GSP that hybridizes as close as possible to the 5’ end; you can also combine the random primers with the GeneRacer Oligo dT primers to increase the chances of obtaining full-length cDNA.
-The cDNA template is a difficult template for PCR: optimize PCR parameters and/or reaction buffer; lower annealing temperature; use 5-10% DMSO in the PCR to help with GC-rich regions; try a high-processivity, high-fidelity PCR enzyme.
-No bands observed after PCR: try different annealing temperatures.
-RNA quality: analyze a sample of your RNA on an agarose gel before starting.

Answer Id: E7333

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Product FAQ

What components does the GeneChip One-Cycle cDNA Synthesis Kit contain?

Answer

The One-Cycle cDNA Synthesis Kit contains all necessary reagents (see the following table) to carry out the cDNA synthesis reaction for thirty samples in a "standard" assay for GeneChip arrays. From total RNA starting materials (1 µg to 15 µg), using this kit, sufficient double-stranded cDNA can be synthesized, purified on the cDNA Cleanup Column, and used as template in the subsequent in vitro transcription labeling reaction.

Component Name: Concentration: Volume: Quantity
T7-Oligo(dT) Primer, 50 µM: 50 µM: 120 µL: 1
5X 1st Strand Reaction Mix: 5X: 120 µL: 1
DTT, 0.1M: 0.1M: 60 µL: 1
dNTP, 10 mM: 10 mM: 120 µL: 1
SµperScript II: 200 U/µL: 60 µL: 1
5X 2nd Strand Reaction Mix 5X: 900 µL: 1
E.coli DNA Ligase: 10 U/µL: 30 µL: 1
E.coli DNA Polymerase I: 10 U/µL: 120 µL: 1
RNase H: 2 U/µL: 30 µL: 1
T4 DNA Polymerase: 5 U/µL: 60 µL: 1
EDTA, 0.5M: 0.5ML: 300 µL: 1
RNase-free Water: n/a: 3.1 mL: 1

Answer Id: E14169

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Product FAQ

What components does the GeneChip Two-Cycle cDNA Synthesis Kit contain?

Answer

The Two-Cycle cDNA Synthesis Kit contains all necessary reagents (see the following table) to carry out the two cycles of cDNA synthesis for thirty samples. Note that the reagents for the intermediary in vitro transcription (IVT) amplification using un-labeled ribonucleotides, between the two cDNA synthesis reactions, is not included in the kit. The Ambion MEGAscript T7 Kit is recommended.

Component Name: Concentration: Volume: Quantity
T7-Oligo(dT) Primer, 50 µM: 50 µM: 120 µL: 1
5X 1st Strand Reaction Mix: 5X: 180 µL: 1
DTT, 0.1M: 0.1M: 90 µ:L 1
dNTP, 10 mM: 10 mM: 200 µL: 1
SuperScript II: 200 U/µL: 60 µL: 1
RNase Inhibitor: 40 U/µL: 45 µL: 1
5X 2nd Strand Reaction Mix: 5X: 900 µL: 1
Random Primers: 3 µg/µL: 60 µL: 1
MgCl2, 1M: 1M: 60 µL: 1
E.coli DNA Polymerase I: 10 U/µL: 200 µL: 1
RNase H: 2 U/µL: 55 µL: 1
T4 DNA Polymerase: 5 U/µL: 60 µL: 1
RNase-free Water: n/a: 9.5 mL: 1

Answer Id: E14170

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Product FAQ

Can Oligo d(T)16 RT primers be used with the TaqMan Human Endogenous Control Plate (P/N 4309199)?

Answer

No. The reverse transcription of total RNA to cDNA must be done with random hexamers or random primers (or a mixture of random and oligo dT primers) because the 18S rRNA assay cannot evaluate poly A+ purified RNA samples as most of the ribosomal RNA has been removed. The plate has been designed to evaluate only total RNA.

Answer Id: E1360

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Product FAQ

How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?

Answer

If amplification products are generated in the control tube/well that contains no reverse transcriptase (i.e., the no-RT control), it may be necessary to eliminate residual genomic DNA from the RNA sample. Use the following protocol to remove genomic DNA from the total RNA preparation.Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions. Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.

Add the following to an autoclaved 0.5 mL microcentrifuge tube on ice:
1.Total RNA, ideally, less than or equal to 1 µg. (See Note 1 below.)
2.1.0 µL of 10X DNase buffer (200 mM Tris, pH 8.3, 500 mM KCl, 20 mM MgCl2).
3.0.1 U-3.0 U of DNase I (RNase-free, Cat. No. 18047019) or 1.0 U Dnase I, Amplification Grade (Cat. No. 18068015. (See Note 2 below.)
4.Bring volume up to 10 µL with DEPC-treated water.
5.Incubate at room temperature for 15 min. (See Note 3 below.)
6.Terminate the reaction by adding 1 µL 25 mM EDTA and heat 10 min at 65 degrees C. (See Note 4 below.)
7.Place on ice for 1 minute.
8.Collect by brief centrifugation. This mixture can be used directly for reverse transcription.

Please note the following:
1.To work with higher quantities of RNA, scale up the entire reaction linearly. Do not exceed 2 µg RNA in the 10 µL reaction. More RNA will increase the viscosity of the solution and prevent the DNAse I from diffusing and finding the DNA.
2.DNAse I, Amplification Grade has been extensively purified to remove trace ribonuclease activities commonly associated with other "RNAse-free" enzyme preparations and does not require the addition of placental RNAse inhibitor.
3.It is important not to exceed the 15 minute incubation time or the room temperature incubation. Higher temperatures and longer times could lead to Mg2+-dependent hydrolysis of the RNA.
4.This procedure requires careful pipetting of all solutions so that the concentration of divalent metal cation (Mg2+) is controlled.
5.Because the DNAse I must be heated to 65 degrees C to inactivate the enzyme, the concentration of free divalent metal ions must be low enough (less than 1 mM) after addition of the EDTA to prevent chemical hydrolysis of the RNA. See references below.
After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg2+ :EDTA. EDTA chelates Mg2+ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg2+. If the reverse transcription buffer does not contain MgCl2, add it to the reaction at a final concentration of 2.5 mM. This results in a net final concentration of approximately 2.25 to 2.5 mM MgCl2.

References on RNA hydrolysis:
Molekulyarnaya Biologiya (1987) 21:1235-1241.
References on the mechanism of hydrolysis by other cations:
Eichorn GL and Butzov JY (1965) Biopolymers 3:79.
Butzov JY and Eichorn GL (1965) Biopolymers 3:95.
Farkas WR (1968) Biochim Biophys Acta 155:401.
The authors of the first paper express the opinion that the mechanism of the nonspecific hydrolysis by cations which proceeds through 2’,3’ cyclic phosphate formation is similar to that of specific hydrolysis such as RNA splicing.

Answer Id: E7322

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Manual / Product Insert

Package Inserts: T7-Oligo (dT) Promoter Primer Kit

Version: 701182 Rev. 3 (06Sep2017)
Catalog #

Product Literature

Data Sheets: T7-Oligo (dT) Promoter Primer Kit

Manual / Product Insert

Manual: Paradise WT-RT User Guide (English )

Version: 29 Jul 2010

Product Literature

Brochure: RNARacer Promotion (Korean)