BeyoRT™ II First Strand cDNA Synthesis Kit with gDNA Eraser

Instruction

1. Removal of genomic DNA from RNA samples
a. Thaw RNA samples and 5X gDNA Eraser Buffer on ice. Thaw 5X RT Buffer, 10X RT Primer Mix, and DEPC-treated Water at room temperature (15-25℃), and immediately place on ice after melting. Mix each solution well prior to use and centrifuge briefly to collect the liquid at the bottom of the tube.
b. RNA denaturation (optional): heat RNA samples at 65℃ for 5 minutes and immediately place on ice, which can effectively improve the reverse transcription of RNAs with secondary structures.
Note: Do not add the gDNA Eraser Buffer at this step.
c. Set up the following reaction on ice. To ensure the accuracy, prepare an appropriate amount of master mix (except RNA) based on the number of reactions, and then dispense into each tube before adding RNA.

*A 20μl reverse transcription reaction requires 10pg to 1μg of Total RNA, and we recommend using 0.5-1μg of Total RNA.
d. Incubate at 37℃ for 2 min on a thermocycler or in a water bath. Then Immediately place the reaction mixture on ice.
2. First-strand cDNA synthesis
a. Referring to the following table, prepare an appropriate amount of 2X Master Mix based on the number of reactions. If the reverse transcription experiment is frequently performed, a mix of 5X RT Buffer and 10X RT Primer Mix at 2:1 can be prepared for frequent use.

* Containing Mg2+ and dNTP
** The mixture of Oligo dT Primer and Random Hexamers
*** Containing RNase Inhibitor
b. Dispense 10μl of the 2X Master Mix into each reaction tube obtained in step 1 and mixed thoroughly.
c. Incubate at 42℃ for 60 min, followed by 10 min at 80℃ to inactivate the reverse transcriptase. Keep the reaction products on ice.
Note: For RNAs with complex secondary structures or high GC content, the reverse transcription temperature can be increased to 50℃ to improve the reverse transcription efficiency.
d. The cDNA products can be used immediately or stored at -20℃ for subsequent experiments such as qPCR and conventional PCR. For long-term storage, it is recommended to store the cDNA at -80℃ in aliquots.
FAQ:
1. The reverse transcription product of total RNA is invisible after electrophoresis.
It is a normal phenomenon, because the amount of RNA template is low, and the amount of reverse transcription products in different size is even lower.
2. No specific product can be amplified from the reverse transcription product, or there is no or delayed signal in qPCR.
a. To exclude the problem of PCR reaction system or reverse transcription product, use gene-specific primers to amplify internal reference genes, such as actin and GAPDH. If reference genes can be amplified, but not the target gene, it indicates primers of target gene are not well designed, the expression of the target gene is too low to be detected, or the reverse transcription efficiency is low.
b. Template RNA may be degraded. The integrity of total RNA can be checked by agarose gel or on-chip electrophoresis. Intact total RNA exhibits sharp, clear 28S and 18S rRNA bands, and the 28S rRNA band should be approximately twice as intense as the 18S rRNA band. A ratio less than 2 indicates the degradation of total RNA and new total RNA should be prepared.
c. RNA samples may contain some components that inhibit the activity of reverse transcriptase. Those contaminants include phenol, SDS, EDTA, guanidine salts, phosphoric acid, pyrophosphoric acid, polyamine, spermidine, etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by BeyoZol (R0011) or Trizol (R0016) usually can meet the requirements of reverse transcription.
d. Insufficient templates for reverse transcription. When DNase I used to remove the residual DNA in RNA sample prior to reverse transcription is subjected to heat-inactivation, EDTA should be added to RNA sample at a final concentration of 2.5mM to protect RNA from degradation under high temperatures. Additionally, to amplify a specific gene, it is necessary to extract RNA from tissues in which the target gene is highly expressed.
e. Inappropriate primer is used for reverse transcription. Random hexamer instead of Oligo(dT)18 should be used for the reverse transcription of bacterial total RNA which does not have poly(A) tails. Gene-specific primers used for reverse transcription must be well designed.
a. The lack of signal or delayed signal in qPCR may be due to the addition of too much reverse transcription product, which will interfere with the PCR reactions. The volume of reverse transcription products added to PCR reactions should be less than 10% of the total volume of PCR reactions, and even less for qPCR reactions.