Recently, Yeasen Biotechnology received a confirmation letter from the U.S. Food and Drug Administration ("FDA") that the DMF data of T7 RNA polymerase submitted by the company has been received by the US FDA, obtained the filing number, and completed the DMF filing.
Figure: FDA has accepted Yeasen's T7 RNA polymerase DMF
If you need to quote the DMF number, please send an email to firstname.lastname@example.org to apply for authorization. After confirming with you, Yeasen Biotechnology will provide you with a DMF authorization letter.
More products of Yeasen Biotechnology are applying for the US FDA DMF filing. Welcome to inquire by email. Email: email@example.com
DMF (Drug Master File) is an archive file submitted by the DMF holder to the FDA, which contains detailed information on the production facilities, technological processes, quality control and the raw materials and packaging materials used in the production, processing, packaging and storage of pharmaceutical products for human use.
The main purpose of DMF holders to submit DMF to FDA is to support various drug applications (one or more clinical investigation applications (IND), innovative drug applications (NDA), generic drug registration (ANDA), export applications, and amendments and additions to the various applications mentioned above). FDA archives the submitted DMF data for review. The DMF holder only needs to provide the user with the authorization letter, authorizing the FDA to conduct a comprehensive examination of the DMF involved when reviewing the user's drug application. Such a DMF system enables drug applicants to directly use the DMF filing number to replace the need to provide specific information on raw materials and excipients during the application process, which saves approval costs, improves approval efficiency, and shortens the registration cycle.
T7 RNA polymerase can use double-stranded DNA containing T7 promoter sequence as a template and NTPs as substrates to synthesize RNA complementary to the single-stranded DNA downstream of the promoter. Therefore, T7 RNA polymerase is commonly used to synthesize long and short transcripts in vitro. Both double-stranded linear blunt-end and 5'-overhang DNA molecules can be used as the transcription template of T7 RNA polymerase, so linear plasmids and PCR products can be used as templates for RNA synthesis in vitro. Yeasen Biotechnology provides you with GMP-grade T7 RNA polymerase, which can produce 100-200 μg of RNA with 1 μg of template input.
1. Low transcript yield
The quality of the template is closely related to the yield. The possible reasons for the significantly lower yield of the experimental group than the control group are as follows:
① There is an inhibitory reaction component in the experimental template.
② The experimental template does not meet the experimental requirements.
① Repurify the template;
② Quantify template and assess 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 sequence will inhibit the reaction. When the transcript is smaller than 100nt, extending the reaction time to 4-8 hours or increasing the amount of template to 2ug can increase the RNA yield.
3. RNA transcript length greater than expected
If electrophoresis shows product bands larger than expected, possible causes:
① The plasmid template may not be completely linearized;
② The sense strand has an overhang at the 3′-end;
③ The RNA has incompletely denatured secondary structures.
① Check whether the template is completely linearized, and if necessary, perform additional linearization;
② Choose a suitable restriction endonuclease to avoid overhanging at the 3'-end of the sense strand, or fill-in with Klenow Fragment or T4 DNA polymerase, and perform transcription after;
③ Use denaturing gel to detect RNA products.
4. RNA transcript length shorter than expected
If electrophoresis shows that the product band is smaller than expected, possible reasons:
① The template contains a termination sequence similar to T7 RNA polymerase;
② The GC content of the template is high.
① Decrease the reaction temperature (for example, 30°C), sometimes lowering the temperature can increase the transcription length, but reduce the yield. Or try different RNA polymerases for transcription;
② If the GC content of the template is high, perform the transcription reaction at 42°C, or add SSB to increase the yield and transcription length.
5. Smearing in electrophoresis of transcripts
Possible reasons for resulting in smearing in the gel electrophoresis:
① The solution was contaminated with RNase during the experimental operation;
② The DNA template was contaminated with RNase.
① Use RNase-free pipette tips and EP tubes during the experiment, wear disposable latex gloves and masks, and prepare all reagents with RNase-free H2O.
② Repurify the template DNA.
UCF.ME® T7 RNA Polymerase GMP-grade（50 U/μL）
UCF.M® T7 RNA Polymerase GMP-grade（50 U/μL）
UCF.M® T7 RNA Polymerase GMP-grade（250 U/μL)