1. Low transcript yield The quality of the template is closely related to the yield. If the yield of the experimental group is significantly lower than the control group, the possible reasons are: ① the experimental template contains inhibitory components; ② The template has something wrong. Suggestions: ① Re-purify the template; ② Determine the template quantification and its integrity; ③ Extend the reaction time; ④ Increase the amount of template input; ⑤ Try other promoters and RNA polymerases. 2. Low yield of short transcripts A short transcription initiation fragment will inhibit the reaction. When the transcription product is less than 100 nt, extending the reaction time to 4-8 hs or increasing the amount of template to 2 μg will increase RNA yield. 3. RNA transcription length is greater than expected If the electrophoresis shows that the product band is larger than the expected size, the possible reasons: ①The plasmid template may not be completely linearized; ②The 3' end of the sense strand has a prominent structure; ③The RNA has a secondary structure that is not completely denatured. Suggestions: ①Check whether the template is completely linearized, and if necessary, perform additional linearization; ②Select a suitable restriction enzyme to avoid 3' overhangs, or use Klenow Fragment /T4 DNA polymerase to complete the transcription before proceeding; ③Use denatured gel to detect RNA products. 4. RNA transcription length is less than expected If the electrophoresis shows that the product band is smaller than the expected size, the possible reasons: ①The template contains a termination sequence similar to T7 RNA polymerase; ②The GC content in the template is high. Suggestions: ①Lower the reaction temperature (for example, 30°C). Sometimes lowering the temperature can increase the transcription length, but it will reduce the yield. Or try different RNA polymerases for transcription; ②If the template GC content is high, use 42℃ to transcript, or add SSB to increase the yield and transcription length. 5. Electrophoresis tailing of transcription products There is a tailing phenomenon during electrophoresis. Possible reasons: ①Contaminated by RNase during experimental operation; ②Contaminated DNA template by RNase. Suggestions: ①Use RNase-free pipette tips and EP tubes, wear disposable latex gloves and masks, and all reagents are prepared with RNase free H2O. ②Re-purify the template DNA.
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