{"id":1227,"date":"2017-06-06T14:50:59","date_gmt":"2017-06-06T14:50:59","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?page_id=1227"},"modified":"2023-03-02T12:30:43","modified_gmt":"2023-03-02T12:30:43","slug":"real-time-pcr","status":"publish","type":"page","link":"https:\/\/www.mybiosource.com\/learn\/assay-learning-center\/real-time-pcr\/","title":{"rendered":"Real-time PCR"},"content":{"rendered":"<p style=\"text-align: left;\">The <span id=\"urn:enhancement-7df25337-aa3e-419f-a550-6be551319548\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">Polymerase<\/span> chain reaction (<span id=\"urn:enhancement-d4d115d2-3d4d-4426-9039-00e3356ae9f8\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span>), one of the most powerful technologies in <span id=\"urn:enhancement-0f4218d1-b5e7-4ff5-9930-6acf2074890f\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/molecular-biology\">molecular biology<\/span>, helps in amplifying or copying specific sequence of <span id=\"urn:enhancement-2c539918-1c21-45cd-a04f-b65b97b42711\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> or complementary template employing oligonucleotides, heat-stable <span id=\"urn:enhancement-4e584aca-f910-48f9-9ac8-db85733d258d\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-786e903e-800d-4010-b3d9-0a9dc32eb054\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> and thermal cycling. With each cycle, the number of target molecules is doubled theoretically and <span id=\"urn:enhancement-63a4a069-6d3c-4df9-8bef-c1c123a9832e\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> is exponentially amplified.<\/p>\n<h3 style=\"text-align: left;\"><strong>Applications<\/strong><\/h3>\n<ul style=\"text-align: left;\">\n<li><span id=\"urn:enhancement-5ba7822c-aca9-49e1-8e93-06ea09625b62\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/gene\">Gene<\/span> <span id=\"urn:enhancement-230725b7-a99f-415c-a6a7-846199aa57d6\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/expression\">Expression<\/span> (mRNA) Analysis<\/li>\n<li>microRNA &amp; Noncoding <span id=\"urn:enhancement-eeb96e5e-3c17-46f4-bcfe-041aed3bf378\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/rna\">RNA<\/span> Analysis<\/li>\n<li><span id=\"urn:enhancement-76fafc0e-74f2-45c4-8746-09e99cdfc1d2\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/genetic-variation\">Genetic variation<\/span> analysis<\/li>\n<li>Protein Analysis<\/li>\n<li>Mutation detection assays<\/li>\n<\/ul>\n<blockquote>\n<p style=\"text-align: left;\"><strong><em><span id=\"urn:enhancement-74a42419-461d-40d7-9ba8-576f3ed2f3ea\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> is the leading tool for detection and <span id=\"urn:enhancement-c3f367fa-ea6a-4194-9cb4-21bce4bb1d9d\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/quantification\">quantification<\/span> of <span id=\"urn:enhancement-081fd809-fd6e-4e5f-919d-349f7072e324\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> and <span id=\"urn:enhancement-f79b9a09-a416-44af-8dc8-2898c7f5afe6\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/rna\">RNA<\/span>. The accuracy and precision for detection within a 2-fold range<\/em><\/strong><\/p>\n<\/blockquote>\n<table class=\" alignleft\" style=\"height: 255px;\" width=\"716\">\n<tbody>\n<tr>\n<td colspan=\"2\" width=\"623\">\n<h3 style=\"text-align: center;\"><strong>Differences: Traditional PCR vs. Real-time PCR<\/strong><\/h3>\n<\/td>\n<\/tr>\n<tr>\n<td width=\"312\"><strong>Traditional <span id=\"urn:enhancement-1ffcfa62-456f-45b8-ac06-c2119607050f\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span><\/strong><\/td>\n<td width=\"312\"><strong>Real-time <span id=\"urn:enhancement-f7dd2bc0-ff6b-4410-b567-c3d43c00fb64\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span><\/strong><\/td>\n<\/tr>\n<tr>\n<td width=\"312\">Detection and <span id=\"urn:enhancement-297a807a-8b12-4233-8e5b-8bc5a59265f6\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/quantification\">quantification<\/span> of the amplified sequence are performed at the end of last cycle.<\/td>\n<td width=\"312\">Detection and <span id=\"urn:enhancement-b3137bc1-622e-464b-a856-a0cdd74bfe4e\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/quantification\">quantification<\/span> of amplified sequence is carried at each cycle.<\/td>\n<\/tr>\n<tr>\n<td width=\"312\">Followed by <span id=\"urn:enhancement-d92e43cd-2698-43d8-b1c9-6d6b22fa2a7e\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/gel-electrophoresis\">gel electrophoresis<\/span> and image analysis for assessment of amplification.<\/td>\n<td width=\"312\">The amount of <span id=\"urn:enhancement-88eb954e-fa09-4c92-96c7-86e02384681e\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> is measured after each cycle using fluorescent dyes. The signal strength is directly proportional to the <span id=\"urn:enhancement-4b6fb0b4-85e3-4bd8-9e84-59ce40530de5\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> product molecules.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<h3><\/h3>\n<h3><\/h3>\n<h3><strong>STEPS<\/strong><\/h3>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-1251 alignleft\" src=\"https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2017\/06\/u111.png\" alt=\"\" width=\"702\" height=\"288\" srcset=\"https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2017\/06\/u111.png 607w, https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2017\/06\/u111-300x123.png 300w, https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2017\/06\/u111-370x152.png 370w, https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2017\/06\/u111-570x234.png 570w\" sizes=\"(max-width: 702px) 100vw, 702px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>The following reactions occupy a central role in real-time <span id=\"urn:enhancement-e5a5b14c-1e3b-49f2-8499-82d5b2949b7a\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span>.<\/p>\n<ol>\n<li style=\"text-align: left;\">Denaturation: Incubation at higher temperature leads to melting of dsDNA into single strands and secondary structure in <span id=\"urn:enhancement-b0fe6191-bfe6-4cee-97f2-00f3afbc675f\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/single-stranded\">single-stranded<\/span> <span id=\"urn:enhancement-1247f02d-1090-4199-833e-2523abe5a8a5\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> is denatured. <span id=\"urn:enhancement-4e0fd162-197a-4b0a-8738-f8c228901299\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-e5e7f269-e31f-4cbc-a5dd-01b135fe8c88\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> can withstand temperature of 95\u00b0C and the timing is increased based on GC concentration.<\/li>\n<li style=\"text-align: left;\">Annealing: Complementary sequences are presented with an opportunity to hybridize at an appropriate temperature, usually by calculating the melting point of the primers involved.<\/li>\n<li style=\"text-align: left;\">Extension: The activity of <span id=\"urn:enhancement-63b7ee36-e221-44fe-9057-c2d6e3376d5e\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-a746e204-3861-4541-99b6-1b73c474893b\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> is optimal at 70\u201372\u00b0C. The extension initiated by primers can occur up to 100 bases per second. When <span id=\"urn:enhancement-479ccfda-e91e-4a7c-9054-6de860ebea94\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> product molecules (amplicon) is small, the extension step is usually combined with annealing at 60\u00b0C temperature.<\/li>\n<\/ol>\n<h3><strong>Reaction components and Parameters<\/strong><\/h3>\n<p><strong><span id=\"urn:enhancement-ece1f83b-ed7f-4f19-ac19-1fd1ba655c25\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-574bbdcb-5745-4691-9eec-477b1efeb867\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span>: <\/strong>The selection of <span id=\"urn:enhancement-ac53fb16-6e22-43f1-819e-9fd23d40fc99\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-a6f3c677-04f6-4b68-9e2b-36761d9be011\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> is critical to the success of assay. Isolated from thermophilic bacterium, the enzymes resist breaking down at higher temperatures and therefore useful in copying <span id=\"urn:enhancement-37d029d5-83f9-4ba6-a804-561a126c87da\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> using a <span id=\"urn:enhancement-9a1927fc-ae88-47cc-ad09-107e307a2663\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> chain reaction. The role of Taq <span id=\"urn:enhancement-0e88448e-b6ea-4da6-adb8-052042d2edc3\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> is to move along the strand of <span id=\"urn:enhancement-f1412793-b348-4bd0-a4f6-dec42021c095\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> and use it as a pattern for assembling a new strand, complementary to the template. For as many as 75 cycles, Taq <span id=\"urn:enhancement-aea152d3-ea7f-425d-8814-4b45ca0f6bc5\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> is useful in <span id=\"urn:enhancement-5f9a8d8b-a7c7-45f8-a306-dc611a69571c\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> to multiply <span id=\"urn:enhancement-a2ec15f6-83dd-4597-82b7-6da74530f907\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> exponentially. Thermo stability of <span id=\"urn:enhancement-c4683742-f5af-4f84-990f-630791f41283\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-221801ca-c74f-4c77-aec1-9ebebda7fda5\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> is comprehensively examined before the selection. Hot start specific antibodies are used to block <span id=\"urn:enhancement-cc254ccf-7693-41ba-ba79-41f90a75958b\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-5e8d0eb4-59db-4a1a-9f82-1f086aa2930d\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> from synthesizing nonspecific product resulting from mispriming. The antibody ensures <span id=\"urn:enhancement-de5366ef-d50d-4964-9890-246ec9e0637b\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-d4f456a9-1b24-43db-8f4c-d0b07cd9d58b\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> is not active during reaction setup and during denaturation.<\/p>\n<p><strong><span id=\"urn:enhancement-6f44aecd-952f-4a34-aff2-db889b76db10\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/reverse-transcriptase\">Reverse transcriptase<\/span><\/strong> transcribes <span id=\"urn:enhancement-5e0c38e7-eb4c-4da8-a2a3-81ef6e193f0f\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/rna\">RNA<\/span> into complementary <span id=\"urn:enhancement-74ddb752-9ae3-4bba-a594-340ee71ce4d7\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span>. Usually, the starting material is <span id=\"urn:enhancement-e57db126-96aa-4962-a766-5213efd070b1\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/rna\">RNA<\/span>. The constituent <span id=\"urn:enhancement-5960b92b-9200-4853-9c9f-c3dcbdb78f5e\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/reverse-transcriptase\">reverse transcriptase<\/span> is as important as <span id=\"urn:enhancement-ec095f0b-7cac-46ca-97dd-5f5f2ae0290e\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-a9935984-5cb5-4ef8-953a-eda1e11625b6\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> as it provides high yields of full-length cDNA and active at higher temperatures. In <span id=\"urn:enhancement-45f8ea78-d2ad-4ecb-b78a-e4f2f7b59834\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/quantification\">quantification<\/span> real-time <span id=\"urn:enhancement-d6d50e0a-8f75-47fe-bb6a-86ad934ca9f6\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span>, <span id=\"urn:enhancement-1ea5a2d7-c2cd-4e05-9357-3c620460fb79\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/reverse-transcriptase\">reverse transcriptase<\/span> active in higher temperature allows use of a <span id=\"urn:enhancement-1b6fdd8a-23a0-48a1-a7fb-ecb88927a66d\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/gene\">gene<\/span> specific primer, thereby increasing specificity and reducing background.<\/p>\n<p><strong>dNTPs<\/strong> (deoxynucleotide triphosphates) is a mixture of four monomeric units such as dATP, dTTP, dCTP and Dgtp. dNTP molecules contains the <span id=\"urn:enhancement-06ae55a3-9105-4d0b-b29e-9c5377c297e8\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> base in a highly energized triphosphate form. These <span id=\"urn:enhancement-25ee78c6-0ec7-47dc-8ccb-4432547cf7d8\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/nucleotides\">nucleotides<\/span> makeup the new DNAs resulting from polymerization reaction as they are added to template strands. Each base is added to <span id=\"urn:enhancement-fe4c90ca-cdc9-459d-af99-ac1fa766bfa9\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> through the <span id=\"urn:enhancement-31496740-1866-4e71-94d8-b87b58423853\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/phosphodiester-bond\">phosphodiester bond<\/span> and a molecule of pyrophosphate.<\/p>\n<p><strong>Magnesium chloride or magnesium sulfate<\/strong> is used at a concentration of 3 mM for most targets and the optimal concentration may vary between 3 and 6mM. Magnesium ions help increasing Taq <span id=\"urn:enhancement-184e2c09-591d-4cc0-b6b2-84c15d6dd27b\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> <span id=\"urn:enhancement-70e012a7-1f36-4c22-a664-de0a015502f1\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> activity at the expense of specificity. Based on the assay, the concentration of magnesium used usually varies.<\/p>\n<p>During replication, dntp&#8217;s gets broken down to dnmp&#8217;s to form <span id=\"urn:enhancement-b8c2edbb-9702-4b8c-bc2f-ff8db7523427\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/phosphodiester-bond\">phosphodiester bond<\/span> between 3&#8242; OH of adjacent nucleotide and 5&#8242; <span id=\"urn:enhancement-92f34df5-697a-4b27-a9f5-ba9b41bf9d00\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/phosphate\">Phosphate<\/span> of the upcoming nucleotide. In this reaction, Mg++ binds to the alpha <span id=\"urn:enhancement-9c0e8d16-da1a-4b3c-9bae-99157ef7f435\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/phosphate\">phosphate<\/span> group of dntp and helps in the removal of beta and gamma <span id=\"urn:enhancement-02c6a1e6-0a81-4def-b9fe-c498af0d3c36\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/phosphate\">Phosphate<\/span> from dntp.<\/p>\n<h3><strong>Template Use<\/strong><\/h3>\n<p>Template nucleic acid use can range between 10 to 1000 per real-time <span id=\"urn:enhancement-bafdb76a-017b-455a-975f-5e43aa429817\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction. This is equivalent to cDNA generated from 1 pg to 100 ng of total <span id=\"urn:enhancement-2260260a-6d8f-4fb0-adb0-00211b78ba6e\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/rna\">RNA<\/span>. Higher use of template nucleic acid may result in greater levels of contaminants that reduces <span id=\"urn:enhancement-f2b15c69-a421-4f94-b4bb-2845f24f833c\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> efficiency. Isolation of mRNA improve the yield of specific cDNAs.<\/p>\n<p>Treating the template with DNase I can significantly help reduce contaminants. Intact <span id=\"urn:enhancement-c413724b-c7f9-4d54-b827-a2ffc9f87112\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/rna\">RNA<\/span> is essential for full-length, high-quality cDNA synthesis and may be important for accurate mRNA <span id=\"urn:enhancement-41cf2b01-b3fd-4f24-bd46-b1b1e5ff4af2\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/quantification\">quantification<\/span>.<\/p>\n<h3><strong>Primer Design Guidelines<\/strong><\/h3>\n<ol>\n<li>Primers should be of length 18-24 <span id=\"urn:enhancement-17d81f88-1f50-41f4-8cd5-6969316e0595\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/nucleotides\">nucleotides<\/span>.<\/li>\n<li>Should be in the range of annealing temperatures.<\/li>\n<li>Specific for the target sequence and be free of internal secondary structure<\/li>\n<li>The primers should necessarily avoid stretches and repeating motifs as it can result inappropriate hybridization.<\/li>\n<li>Primers should have GC content of 50% and compatible melting temperatures.<\/li>\n<\/ol>\n<h3><strong>Primer Design Software<\/strong><\/h3>\n<p style=\"text-align: left;\">Using primer design software programs ensure that primers are specific for targets and are free of secondary structure. Also, it helps in avoiding complementary hybridization at 3\u0374 ends within each primer. Some of the notable primer design software programs are Invitrogen, OligoPerfect and Primer Express. These software programs are sophisticated enough to automate analysis of sequence and design primers.<\/p>\n<h3 style=\"text-align: left;\"><strong>Analysis<\/strong><\/h3>\n<p>This section covers the definitions used in RT <span id=\"urn:enhancement-aab71938-f9fa-4e31-8fa2-ffcdf20524d1\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> analysis.<\/p>\n<p><strong>Baseline<\/strong> refers to the signal level during initial cycle of <span id=\"urn:enhancement-51aee9ce-082a-4ff1-8f96-eb67f0ecf731\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span>, between cycle 3 and 15. During these cycles, there are little changes in fluorescent signal. The little changes is attributed to the background or noise of the reaction. The baseline is usually moderated to allow accuracy of the threshold cycle.<\/p>\n<p><strong>Threshold<\/strong> is the level of signal that reflect statistically significant rise over the calculated baseline signal. This is set to distinguish amplification signal from the background. Mostly, software sets the threshold at 10 times the standard deviation of value of the baseline.<\/p>\n<p><strong>Threshold cycle<\/strong> is the cycle number at which fluorescent signal crosses the threshold. The cycle number is inversely proportional to the starting amount of target. It is useful in calculating the initial <span id=\"urn:enhancement-82324477-d642-4dcd-9c95-d952d4956e03\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-strand\">DNA<\/span> copy number.<\/p>\n<p><strong>The correlation coefficient<\/strong> is a measure of how well the data fit the standard curve.<\/p>\n<p><strong>Dynamic range<\/strong> is the range over which an increase in starting material concentration results in a corresponding increase in amplification product.<\/p>\n<h3><strong>Advantages<\/strong><\/h3>\n<ul>\n<li>Ability to monitor individual reaction as they happen in real time.<\/li>\n<li>Measuring the quantity of amplicon at each cycle and thereby accurate <span id=\"urn:enhancement-07f742bb-9f15-4c48-8aed-cf591a9533b3\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/quantification\">quantification<\/span> of starting material in samples.<\/li>\n<li>Dynamic detection<\/li>\n<\/ul>\n<p><strong>\u00a0<\/strong><\/p>\n","protected":false},"excerpt":{"rendered":"<p>The Polymerase chain reaction (PCR), one of the most powerful technologies in molecular biology, helps in amplifying or copying specific sequence of DNA or complementary template employing oligonucleotides, heat-stable DNA polymerase and thermal cycling. With each cycle, the number of target molecules is doubled theoretically and DNA is exponentially amplified. Applications Gene Expression (mRNA) Analysis [&hellip;]<\/p>\n","protected":false},"author":2,"featured_media":0,"parent":1220,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"class_list":["post-1227","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1227","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/comments?post=1227"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1227\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1220"}],"wp:attachment":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/media?parent=1227"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}