Isolation of functional total RNA from Tilia cordata leaves and pollen
| Autor: | Jana Ognjenovic, Omar Ziyad Tantoush, Jelena Vukmirica, Ratko M. Jankov, Ćirković Tanja Veličković |
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| Rok vydání: | 2012 |
| Předmět: |
Tilia cordata
RT-PCR 010402 general chemistry medicine.disease_cause 01 natural sciences lcsh:Chemistry Research Potential Complementary DNA Pollen Botany medicine 2. Zero hunger chemistry.chemical_classification biology Chemistry food and beverages General Chemistry Isolation (microbiology) biology.organism_classification 0104 chemical sciences Reverse transcription polymerase chain reaction genomic DNA total RNA Enzyme Real-time polymerase chain reaction lcsh:QD1-999 pollen leaves cDNA |
| Zdroj: | Journal of the Serbian Chemical Society Journal of the Serbian Chemical Society, Vol 77, Iss 8, Pp 1003-1012 (2012) |
| ISSN: | 1820-7421 0352-5139 |
| DOI: | 10.2298/jsc111130018o |
| Popis: | The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol (TM) preparation of the total RNA. The total RNA isolated using TRIzol (TM) was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first-and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8 +/- 7.9 mu g g(-1) leaves; 25.7 +/- 1.1 mu g g(-1) pollen) could be obtained and was suitable for application in further molecular biology studies. Uspostavljeni su uslovi za izolovanje ukupne RNK iz lišća i polena evropske lipe (Tilia cordata). Korišćenjem komercijalno dostupnog pribora za izolovanje RNK iz biljaka izolovana je čista ukupna RNK iz lišća lipe, dok je korišćenjem iste metode dobijena degradirana RNK iz polena lipe. Uspešno izolovanje RNK iz lišća i polena je dobijeno korišćenjem TRIzol reagensa. RNK izolovana ovim metodom je kontaminirana genomskom DNK, koja je uspešno eliminisana korišćenjem enzima DNaze. Dalje su optimizovani i uslovi uklanjanja genomske DNK pomoću DNaze. Izolovana ukupna RNK iz oba izvora je dalje uspešno iskorišćena za sintezu prvog i drugog lanca klonske DNK, kao i u reverzno-transkriptivnoj PCR reakciji, dokazujući time da je korišćenjem ovog metoda izolovana funkcionalna ukupna RNK. U zaključku, dobijena je čista i funkcionalna RNK iz lišća i polena T. cordata (27,8±7,9 μg g-1 lišća; 25,7±1,1 μg g-1 polena) koja se može koristiti u daljim molekularno-biološkim istraživanjima. |
| Databáze: | OpenAIRE |
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