American Journal of Plant Sciences
Vol.5 No.13(2014), Article ID:47467,9 pages DOI:10.4236/ajps.2014.513221

An Efficient Protocol for Total RNA Isolation from Healthy and Stressed Tissues of Mulberry (Morus sp.) and Other Species

Figure 1. Denaturing agarose gel stained with ethidium bromide showing total RNA isolated from leaf tissues of mulberry using different RNA isolation methods (Logemann et al. (1987), Gasic et al. (2004), Salzman et al. (1999), TRIzol® (Invitrogen), RNeasy®(Qiagen) and modified protocol). Starting material and the volume of the DEPC-treated water used to dissolve RNA were kept constant and 2 μl total RNA was loaded for comparison.

(Figure 1). The reaction components included RNase inhibitors, chelator (EDTA), and protein denaturants (SDS and β-mercaptoethanol). We used PVPP instead of PVP in the standardized protocol. Addition of PVPP in nucleic acid extraction protocols was shown to be very effective for preventing tissue browning [5] [18] . Further, pH of the solution was maintained in the acidic range to allow efficient and preferable partitioning of RNA in the aqueous phase leaving DNA in the phenolic phase [19] .

The modified protocol yielded a white, water soluble RNA precipitate from different tissues of mulberry. Denaturing agarose gel stained with ethidium bromide (EtBr) indicated intact RNA as there was clear distinct bands of 28 and 18S rRNA (Figure 1). About 520.00 ± 2.60 μg total RNA per gram of fresh leaf tissue was harvested from mulberry and the ratios of 260/280 and 260/230 were 2.02 ± 0.02 and 2.06 ± 0.02, respectively suggesting low quantities of interfering compounds such as polysaccharides, polyphenols and proteins (Table 2). The method developed was further used to extract RNA from different plant tissues (root, stem, inflorescence and immature fruits) of mulberry. As shown in the Figure 2, and Table 3, good quality RNA was extracted from these tissues, signifying that the proposed protocol could be useful for different tissue types. Similarly, the proposed protocol yielded satisfactory results for RNA extraction from plant species, such as cardamom, papaya and rice. The total RNA isolated from leaf tissues contained intact 28 and 18S rRNA signatures with high quantity (Figure 3, Table 4). We believe that this protocol could be useful for extracting RNA from many other species having metabolites that act as nucleic acids interfering molecules.

Many studies on stress biology require extraction of high quality RNA and many protocols failed to yield quality RNA in sufficient quantities [20] [21] . However, using this standardised protocol, we could isolate quality RNA in sufficient quantities from mulberry leaf tissues subjected to dehydration and salinity stresses. We isolated high quality RNA from stressed leaf tissues (Figure 4), and the average yield of total RNA was 348.83 ± 12.20 and 293.78 ± 7.20 μg/g of fresh weight under dehydration and salinity stresses, respectively (Table 4).


Radha Sivarajan Sajeevan1,2, Manchanahally Byrappa Shivanna2, Karaba N. Nataraja1*