{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T19:00:48Z","timestamp":1787166048849,"version":"build-2736575974"},"reference-count":6,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T00:00:00Z","timestamp":1783728000000},"content-version":"vor","delay-in-days":10,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:00:00Z","timestamp":1782864000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The extraction of extracellular RNA from human serum is challenging due to its low abundance, high degree of fragmentation, and susceptibility to rapid degradation. These limitations are further compounded in pediatric research, where low blood sample volume is a major, frequent constraint, necessitating highly sensitive and efficient RNA extraction methods suitable for minimal\u2010input samples. Existing protocols use TRIzol or commercial kits, which are primarily optimized for the enrichment of small RNAs rather than for the comprehensive recovery of extracellular RNA. Here, we present an optimized protocol for efficient isolation of extracellular RNA from human serum, using a modified TRIzol\u2010based method following chloroform phase separation. We processed serum samples from pediatric patients using TRI\u2010Reagent\u2010based lysis and chloroform phase separation, and then compared the results of subsequent isopropanol precipitation (protocol 1) and silica\u2010column\u2010based purification (protocol 2). Protocol 2 significantly improved RNA yield (5\u2010fold), purity (\n                    <jats:italic>A<\/jats:italic>\n                    <jats:sub>260<\/jats:sub>\n                    \/\n                    <jats:italic>A<\/jats:italic>\n                    <jats:sub>280<\/jats:sub>\n                    and\n                    <jats:italic>A<\/jats:italic>\n                    <jats:sub>260<\/jats:sub>\n                    \/\n                    <jats:italic>A<\/jats:italic>\n                    <jats:sub>230<\/jats:sub>\n                    ratios), and integrity compared to protocol 1, as confirmed by agarose gel electrophoresis. The extracted RNA was successfully utilized for cDNA synthesis and qRT\u2010PCR amplification of the human\n                    <jats:italic>CRBN<\/jats:italic>\n                    gene, demonstrating its suitability for downstream applications. Protocol 2 provides a rapid, cost\u2010effective, and robust approach for isolating extracellular RNA from serum, validated primarily for RT\u2010PCR\u2010based applications, yielding RNA of improved quantity and purity suitable for biomarker analysis and gene expression studies from minimal\u2010input clinical samples. \u00a9 2026 Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol<\/jats:bold>\n                    : Optimized isolation of extracellular RNA from human serum using TRIzol\u2010based lysis and chloroform phase separation\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 1<\/jats:bold>\n                    : Isopropanol\u2010precipitation\u2010based recovery of extracellular RNA from aqueous phase\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 2<\/jats:bold>\n                    : Silica\u2010column\u2010based purification of extracellular RNA for enhanced yield and purity\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 3<\/jats:bold>\n                    : cDNA synthesis and qRT\u2010PCR analysis of extracellular RNA.\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70422","type":"journal-article","created":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T14:10:16Z","timestamp":1783779016000},"source":"Crossref","is-referenced-by-count":1,"title":["An Optimized and Improved Protocol for Efficient Isolation of Extracellular RNA from Human Serum"],"prefix":"10.1002","volume":"6","author":[{"given":"Rahul","family":"Patel","sequence":"first","affiliation":[{"name":"Department of Pediatrics All India Institute of Medical Sciences  Raebareli Uttar Pradesh India"},{"name":"Department of Biochemistry All India Institute of Medical Sciences  Raebareli Uttar Pradesh India"},{"name":"Authors contributed equally"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8133-013X","authenticated-orcid":false,"given":"Namita","family":"Mishra","sequence":"additional","affiliation":[{"name":"Department of Pediatrics All India Institute of Medical Sciences  Raebareli Uttar Pradesh India"},{"name":"Authors contributed equally"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rasna","family":"Gupta","sequence":"additional","affiliation":[{"name":"Department of Pediatrics All India Institute of Medical Sciences  Raebareli Uttar Pradesh India"},{"name":"Authors contributed equally"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0503-2322","authenticated-orcid":false,"given":"Ankit","family":"Gupta","sequence":"additional","affiliation":[{"name":"Department of Biochemistry All India Institute of Medical Sciences  Raebareli Uttar Pradesh India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,11]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.53446\/actamednicomedia.1628412"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u20104939\u20107652\u20102_5"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.3791\/51443"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0082753"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot5439"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb2210"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70422","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70422","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70422","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:33:41Z","timestamp":1785854021000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70422"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7]]},"references-count":6,"journal-issue":{"issue":"7","published-print":{"date-parts":[[2026,7]]}},"alternative-id":["10.1002\/cpz1.70422"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70422","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7]]},"article-number":"e70422"}}