双链短RNA[dsRNA]合成方法（How do you synthesize your dsRNA）
互联网
We routinely produce dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends (I. Primer Designed dsRNA). It is also possible to produce dsRNA using PCR generated DNA templates containing either the T7 & SP6 or the T7 & T3 promoters on either end (II. dsRNA Fom Clones). This method is less efficient, especially when working on a large scale.
~undefined~2~1~0~0~0~0All_work_should_be_done_in_a_sterile~E_RNase_free_environment~E_using_only_sterile~E_RNase_free_solutions_and_materials~E_and_while_wearing_gloves_do_reduce_contamination.~0~0~0~K~Hp~M~2~1~0~0~0~Kp~M~2~1~0~0~0~0~Ka_name~L~4I.~4~M~2~1~0~0~0~0~0I._Primer_Designed_dsRNA~0~0~0~0~2~1~0~0~0~K~Ha~M~K~Hp~M~Ka_name~L~4I.~4~M~2~1~0~0~0~K~Ha~M~Kp~M~Ka_name~L~4I.~4~M~2~1~0~0~0~0~K~Ha~M~Ka_name~L~4I.A.~4~M~2~1~0~0~0~0~0A._Template_Selection~0~0~0~0~2~1~0~0~0~K~Ha~M~K~Hp~M~Ka_name~L~4I.A.~4~M~2~1~0~0~0~Kp~MTemplates_can_be_generated_by_PCR_on_cDNA_~Aincluding_ESTs_from_BDGP~B~E_genomic_DNA~E_or_first_strand_RT~FcDNA._Most_of_the_dsRNA_should_correspond_to_exons_but_dsRNA_with_two_or_more_exons_interrupted_by_introns_will_also_work_well._We_generally_aim_for_~X500_bp_products_although_RNAi_with_products_ranging_from_150~F3000bp_have_been_shown_to_work._The_target_sequence_should_not_contain_complete_21~Fmer_homology_to_other_genes_or_your_dsRNA_could_be_non~Fspecific._One_case_has_been_seen_where_the_template_contained_one_mismatch_in_a_21~Fmer_identical_stretch_to_another_gene~E_and_still_only_the_level_of_the_intended_target_was_reduced~E_while_the_close_homologue_was_unaffected._Since_the_targeted_region_within_a_transcript_may_influence_the_success_of_RNAi_~E_the_safest_approach_is_to_use_dsRNA_corresponding_to_the_5~4_or_3~4_UTR._It_is_recommended_to_check_the_various_sequence_sources_~APublications~E_NCBI_and_BDGP~E_genomic_and_ESTs~B_to_confirm_the_primary_sequence_of_a_given_gene.~K~Hp~M~2~1~0~0~0~Kp~M~2~1~0~0~0~0We_suggest_using_the_Primer3_website_~A~2~1~0~0~0~0~2~1~0~0~0~0~0http~I~H~Hwww~Fgenome.wi.mit.edu~Hcgi~Fbin~Hprimer~Hprimer3~Swww.cgi~2~1~0~0~0~0~2~1~0~0~0~0~B_for_designing_primers._Complementary_sequences_should_be_20~F24_nt~E_the_Tm_range_between_59~NC_~F_61~NC_and_optimize_against_self~Fannealing_by_setting_the_Max_Complementarity_to_5_and_Max_3~4_Complementarity_to_1._After_choosing_the_primer_sequence~E_add_the_T7_promoter_sequence_~ATAATACGACTCACTATAGGGAGA~B_to_the_5~4_end.~0~0~0~K~Hp~M~2~1~0~0~0~K~Ha~M~Kp~M~Ka_name~L~4I.A.~4~M~2~1~0~0~0~0~K~Ha~M~Ka_name~L~4I.B.~4~M~2~1~0~0~0~0~0B._PCR~0~0~0~0~2~1~0~0~0~K~Ha~M~K~Hp~M~Ka_name~L~4I.B.~4~M~2~1~0~0~0~Kp~MPerform_a_standard_50_or_100µl PCR reaction using your selected primer sequences to amplify the region of interest. Use 1-2 µl of a 10µM primer stock, 100-200 ng DNA as template and DNA polymerase. We have successfully used GeneChoice Taq (PGC Scientifics #62-6086-01), TaqPlus (Stratagene #600210), and Herculase (Stratagene #NC9690330) in the following PCR reaction conditions:
- 94 o C - 3 min.
- 94 o C - 45 sec.
- 57 o C - 30 sec.
- 72 o C - 45 sec.
- steps 2-4, 30x
- 72 o C - 10 min.li
Check the PCR results to ensure that you have a band of the expected size. I run 4ul in a 1% agarose gel and use the small combs. I also us a 250bp ladder.
We use the Ambion MEGAscript T7 kit (Cat.# : 1334) for the transcription reaction. Follow the Ambion MEGAscript kit protocol for Transcription Reaction Assembly and use 5 µl of PCR template per 20 µl reaction. It is not necessary to purify the PCR template before transcription. We incubate the reaction in a heat block or thermocycler for 4 hours. Following incubation, we remove the DNA template with the DNase. Transcription and annealing occur simultaneously and no additional step is required to anneal the two complementary. If you want more dsRNA, scale up the reaction.
Confirm that the dsRNA product is the correct size using a 1% agarose gel with TBE or TAE buffer and load only 0.5 µl.
