{"id":2193,"date":"2015-04-28T08:12:50","date_gmt":"2015-04-28T08:12:50","guid":{"rendered":"http:\/\/www.neb-online.de\/?page_id=2193"},"modified":"2023-06-23T11:02:03","modified_gmt":"2023-06-23T09:02:03","slug":"in-vitro-rna-synthesis","status":"publish","type":"page","link":"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/","title":{"rendered":"In vitro RNA Synthesis"},"content":{"rendered":"

[vc_row][vc_column][vc_basic_grid post_type=”post” max_items=”10″ element_width=”12″ gap=”15″ item=”4123″ taxonomies=”131″ grid_id=”vc_gid:1687510891164-305f9337-f15b-4″][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

Background<\/h2>\n

In vitro<\/em>\u00a0synthesis<\/a> of single-stranded RNA molecules is a routine laboratory procedure that is essential to the rapidly developing field of RNA research. This technique is versatile in that it allows the researcher to tailor synthesis, and introduce modifications that produce a transcript that is optimal for use in downstream applications. Though the ways in which RNA can be produced are countless, the same basic procedure is followed in all synthesis protocols.[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=”black”][\/vc_column][\/vc_row][vc_row css=”.vc_custom_1519721283817{margin-right: -1px !important;margin-left: -1px !important;background-color: #f4f1e7 !important;}”][vc_column width=”1\/2″ css=”.vc_custom_1519721276790{border-right-width: 5px !important;border-right-color: #ffffff !important;border-right-style: solid !important;}”][vc_row_inner][vc_column_inner width=”2\/3″][vc_column_text css=”.vc_custom_1519721419578{margin-bottom: 55px !important;}”]<\/p>\n

Our free brochure:<\/strong>
\nRNA Synthesis \u2013
\nFrom Template to Transcript<\/strong><\/span><\/p>\n

[\/vc_column_text]Order Brochure<\/a>[\/vc_column_inner][vc_column_inner width=”1\/3″][vc_single_image image=”1373″ img_size=”full” alignment=”center” style=”vc_box_shadow”][\/vc_column_inner][\/vc_row_inner][\/vc_column][vc_column width=”1\/2″][vc_row_inner][vc_column_inner width=”2\/3″][vc_column_text]<\/p>\n

Feature Article:<\/strong>
\nMinding your caps and tails \u2013 considerations for functional mRNA synthesis<\/strong><\/span><\/p>\n

[\/vc_column_text]Download PDF<\/a>[\/vc_column_inner][vc_column_inner width=”1\/3″][vc_single_image image=”45409″ img_size=”full” alignment=”center” style=”vc_box_shadow” onclick=”custom_link” img_link_target=”_blank” link=”https:\/\/www.neb.com\/en\/-\/media\/nebus\/files\/expressions\/nebexpressions_feature_handout_issueiii_2021_rna_capstails.pdf”][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=”black”][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

Workflow\u00a0Details<\/h2>\n

Template Generation<\/h3>\n

The first step in\u00a0in vitro<\/em>\u00a0RNA synthesis is to prepare the DNA template corresponding to the sequence of interest. To allow run off transcription, plasmid DNA template is generally linearized with a\u00a0restriction enzyme<\/a>. In addition to plasmid DNA, PCR products and synthetic oligonucleotides can be used as templates for transcription reactions. We recommend Q5 High-Fidelity DNA Polymerase<\/a>\u00a0if you plan to use a PCR product as template.<\/p>\n

In vitro<\/em>\u00a0 Synthesis<\/h3>\n

The template DNA is transcribed by a T7, T3 or SP6 RNA phage polymerase in the presence of ribonucleoside triphosphates (rNTPs). The polymerase traverses the template strand and uses base pairing with the DNA to synthesize a complementary RNA strand (using uracil in the place of thymine). The RNA polymerase travels from the 3′ \u2192 5′ end of the DNA template strand, to produce an RNA molecule in the 5′ \u2192 3′ direction.<\/p>\n

The\u00a0HiScribe\u2122 T7 Quick High Yield RNA Synthesis Kit<\/a>\u00a0is designed for quick set-up and production of large amounts of RNA in vitro. The reaction can be set up conveniently by combining the NTP buffer mix, T7 RNA Polymerase mix and a suitable DNA template. The kit also allows for capped RNA or dye-labeled RNA synthesis by incorporation of cap analog (ARCA<\/a>) or dye-modified nucleotides. The\u00a0HiScribe\u2122 T7 High Yield RNA Synthesis Kit<\/a>\u00a0is an extremely flexible system for\u00a0in vitro<\/em>\u00a0transcription of RNA using T7 RNA Polymerase. The kit allows for synthesis many kinds of RNA including internally labeled and co-transcriptionally capped<\/p>\n

Combined\u00a0RNA Synthesis and Modification<\/h3>\n

For combined RNA synthesis and modification NEB offers the\u00a0HiScribe\u2122 T7 ARCA mRNA Kit<\/a>\u00a0and the\u00a0HiScribe\u2122 T7 ARCA mRNA Kit (with tailing)<\/a>.
\nBoth kits include reagents need for RNA synthesis, modification and clean-up.transcripts.<\/p>\n

RNA Modification<\/span><\/h3>\n

mRNAs synthesized\u00a0in vivo<\/em> by eukaryotic organisms are co-transcriptionally modified by the addition of a a cap structure, which consists of a 5′ 7-methyl guanosine residue that protects the transcript from nuclease digestion and promotes translation. Additionally, eukaryotic mRNAs are 3\u2019-polyadenylated by a template independent polymerase. The poly(A) tail also functions in mRNA stability and translational regulation.<\/p>\n

For experiments where RNA will be introduced into cells by transfection or microinjection,\u00a0in vitro<\/em> transcripts can be modified to mimic mRNA by using the Faustovirus Capping Enyzme (FCE)<\/a>. Faustovirus Capping Enzyme (FCE) is a single-subunit enzyme that catalyzes the addition of N7<\/sup>-methylguanosine cap (m7<\/sup>G) to the 5\u2032 end of triphosphorylated and diphosphorylated transcripts, producing Cap-0 RNA. <\/span>FCE retains significant capping activity at low temperatures and tolerates reaction temperatures up to 55\u00b0C. FCE offers best value with improved capping efficiency, even on difficult substrates.<\/p>\n

Moreover, we offer mRNA capping enzymes from vaccinia virus<\/a>\u00a0to add cap structures, and\u00a0E. coli<\/em>\u00a0poly(A) polymerase<\/a>\u00a0to add poly(A) tails.<\/p>\n

Alternatively, cap structure analogs, such as (m7G(5′)ppp(5′)G)<\/a>, may be used to co-transcriptionally cap RNA transcripts during synthesis.<\/p>\n

Synthesized RNA can be used in a number of applications including RNA structure and function studies, ribozyme biochemistry, probes for RNase protection assays and hybridization based blots, anti-sense RNA and RNAi experiments, microarray analysis, microinjection,\u00a0in vitro<\/em>\u00a0translation, and RNA vaccines.<\/p>\n

Please use our\u00a0RNA Cap Analog Selection Chart<\/a>\u00a0to find a suitable Cap Analog for your experiment.<\/p>\n

RNA Clean-Up<\/h3>\n

After successful in vitro<\/em> transcription the template is degraded either with DNaseI<\/a> or DNaseI-XT<\/a> (recommended), which is a superior DNaseI variant and active at higher salt concentrations typical for IVT reactions. mRNAs can be affinity purified with Oligo(dT)Beads<\/a>.[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=”black”][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

mRNA synthesis workflow example & available NEB products<\/h2>\n

(Click on the respective box\/product for more product information)<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Template Generation<\/th>\nIn vitro<\/em> Transcription<\/th>\nRNA Capping<\/th>\nPoly(A) Tailing<\/th>\nRNA Purification<\/th>\n<\/tr>\n
Q5 High-Fidelity DNA Polymerase<\/a><\/td>\nHiScribe T7 mRNA Kit with CleanCap Reagent AG<\/a><\/td>\nMonarch RNA Cleanup Kit (10\u00a0\u00b5g)<\/a><\/td>\n<\/tr>\n
HiScribe T7 ARCA mRNA Synthesis Kit (with tailing)<\/a><\/td>\n<\/tr>\n
dNTP solution mixes<\/a><\/td>\nHiScribe T7 ARCA mRNA Synthesis Kit<\/a><\/td>\nE.\u00a0coli<\/em> Poly(A) Polymerase<\/a><\/td>\nMonarch RNA Cleanup Kit (50\u00a0\u00b5g)<\/a><\/td>\n<\/tr>\n
BspQI<\/a><\/td>\nHiScribe T7 High Yield RNA Synthesis Components<\/a><\/td>\nFaustovirus
\nCapping Enzyme<\/a><\/td>\n		<\/td>\nMonarch RNA Cleanup Kit (500\u00a0\u00b5g)<\/a><\/td>\n<\/tr>\n
DNA Assembly<\/p>\nHiScribe T7 Quick High Yield RNA Synthesis Kit<\/a><\/td>\nVaccinia Capping System<\/a><\/td>\n<\/td>\n<\/tr>\n
HiScribe SP6 High Yield RNA Synthesis Kit<\/a><\/td>\nmRNA Cap 2\u00b4-O-Methyltranferase<\/a><\/td>\n<\/td>\n<\/tr>\n
T3 RNA Polymerases<\/a><\/td>\nARCA and other mRNA cap analogs<\/a><\/td>\n<\/tr>\n
SP6 RNA Polymerases<\/a><\/td>\n<\/td>\n<\/tr>\n
T7 RNA Polymerase<\/a><\/td>\n<\/tr>\n
Hi-T7 RNA Polymerase<\/a><\/td>\n<\/tr>\n
Companion Products<\/strong><\/td>\nCompanion Products<\/strong><\/td>\n<\/tr>\n
RNase inhibitor (Murine)<\/a><\/td>\nMonarch RNA Cleanup Binding Buffer<\/a><\/td>\n<\/tr>\n
RNase Inhibitor (Human Placental)<\/a><\/td>\nMonarch RNA Cleanup Wash Buffer<\/a><\/td>\n<\/tr>\n
Pyrophosphatase, Inorganic (E.\u00a0coli<\/em>)<\/a><\/td>\nNuclease-free Water<\/a><\/td>\n<\/tr>\n
Pyrophosphatase, Inorganic (Yeast)<\/a><\/td>\n<\/td>\n<\/tr>\n
DNase I-XT<\/a><\/td>\n<\/tr>\n
NTPs<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

[\/vc_column_text][\/vc_column][\/vc_row]<\/p>\n<\/div>","protected":false},"excerpt":{"rendered":"

[vc_row][vc_column][vc_basic_grid post_type=”post” max_items=”10″ element_width=”12″ gap=”15″ item=”4123″ taxonomies=”131″ grid_id=”vc_gid:1687510891164-305f9337-f15b-4″][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text] Background In vitro\u00a0synthesis of single-stranded RNA molecules is a routine laboratory procedure that is essential to the rapidly developing field of RNA research. This technique is versatile in that it allows the researcher to tailor synthesis, and introduce modifications that produce a transcript that is optimal for… Read more »<\/a><\/p>\n","protected":false},"author":2,"featured_media":40787,"parent":2191,"menu_order":0,"comment_status":"open","ping_status":"closed","template":"page-sidebar.php","meta":{"footnotes":""},"class_list":["post-2193","page","type-page","status-publish","has-post-thumbnail","hentry"],"yoast_head":"\nIn vitro RNA Synthesis - New England Biolabs GmbH<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"In vitro RNA Synthesis - New England Biolabs GmbH\" \/>\n<meta property=\"og:description\" content=\"[vc_row][vc_column][vc_basic_grid post_type=”post” max_items=”10″ element_width=”12″ gap=”15″ item=”4123″ taxonomies=”131″ grid_id=”vc_gid:1687510891164-305f9337-f15b-4″][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text] Background In vitro\u00a0synthesis of single-stranded RNA molecules is a routine laboratory procedure that is essential to the rapidly developing field of RNA research. This technique is versatile in that it allows the researcher to tailor synthesis, and introduce modifications that produce a transcript that is optimal for... Read more »\" \/>\n<meta property=\"og:url\" content=\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/\" \/>\n<meta property=\"og:site_name\" content=\"New England Biolabs GmbH\" \/>\n<meta property=\"article:modified_time\" content=\"2023-06-23T09:02:03+00:00\" \/>\n<meta property=\"og:image\" content=\"https:\/\/www.neb-online.de\/wp-content\/uploads\/2015\/04\/NEB_rnabiologie_Header_x760px.jpg\" \/>\n\t<meta property=\"og:image:width\" content=\"760\" \/>\n\t<meta property=\"og:image:height\" content=\"200\" \/>\n\t<meta property=\"og:image:type\" content=\"image\/jpeg\" \/>\n<meta name=\"twitter:label1\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data1\" content=\"6 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\/\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/\",\"url\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/\",\"name\":\"In vitro RNA Synthesis - New England Biolabs GmbH\",\"isPartOf\":{\"@id\":\"https:\/\/www.neb-online.de\/#website\"},\"primaryImageOfPage\":{\"@id\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/#primaryimage\"},\"image\":{\"@id\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/#primaryimage\"},\"thumbnailUrl\":\"https:\/\/www.neb-online.de\/wp-content\/uploads\/2015\/04\/NEB_rnabiologie_Header_x760px.jpg\",\"datePublished\":\"2015-04-28T08:12:50+00:00\",\"dateModified\":\"2023-06-23T09:02:03+00:00\",\"breadcrumb\":{\"@id\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/#breadcrumb\"},\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/\"]}]},{\"@type\":\"ImageObject\",\"inLanguage\":\"en-US\",\"@id\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/#primaryimage\",\"url\":\"https:\/\/www.neb-online.de\/wp-content\/uploads\/2015\/04\/NEB_rnabiologie_Header_x760px.jpg\",\"contentUrl\":\"https:\/\/www.neb-online.de\/wp-content\/uploads\/2015\/04\/NEB_rnabiologie_Header_x760px.jpg\",\"width\":760,\"height\":200},{\"@type\":\"BreadcrumbList\",\"@id\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Home\",\"item\":\"https:\/\/www.neb-online.de\/en\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"RNA Biology\",\"item\":\"https:\/\/www.neb-online.de\/en\/rna-biology\/\"},{\"@type\":\"ListItem\",\"position\":3,\"name\":\"In vitro RNA Synthesis\"}]},{\"@type\":\"WebSite\",\"@id\":\"https:\/\/www.neb-online.de\/#website\",\"url\":\"https:\/\/www.neb-online.de\/\",\"name\":\"New England Biolabs GmbH\",\"description\":\"Enabling Technologies in the Life Sciences\",\"potentialAction\":[{\"@type\":\"SearchAction\",\"target\":{\"@type\":\"EntryPoint\",\"urlTemplate\":\"https:\/\/www.neb-online.de\/?s={search_term_string}\"},\"query-input\":{\"@type\":\"PropertyValueSpecification\",\"valueRequired\":true,\"valueName\":\"search_term_string\"}}],\"inLanguage\":\"en-US\"}]}<\/script>\n<!-- \/ Yoast SEO plugin. -->","yoast_head_json":{"title":"In vitro RNA Synthesis - New England Biolabs GmbH","robots":{"index":"index","follow":"follow","max-snippet":"max-snippet:-1","max-image-preview":"max-image-preview:large","max-video-preview":"max-video-preview:-1"},"canonical":"https:\/\/www.neb-online.de\/en\/rna-biology\/in-vitro-rna-synthesis\/","og_locale":"en_US","og_type":"article","og_title":"In vitro RNA Synthesis - New England Biolabs GmbH","og_description":"[vc_row][vc_column][vc_basic_grid post_type=”post” max_items=”10″ element_width=”12″ gap=”15″ item=”4123″ taxonomies=”131″ grid_id=”vc_gid:1687510891164-305f9337-f15b-4″][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text] Background In vitro\u00a0synthesis of single-stranded RNA molecules is a routine laboratory procedure that is essential to the rapidly developing field of RNA research. 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