Background

Paulownia elongata is a widely distributed tree that belongs to the family of Paulowniaceae (Zhu et al., 1986). It is known for its high adaptability and rapid growth rate (Chaires et al., 2017). Paulownia woods are gaining demands from all over the world due to their high stability, low thermal conductivity, decay and rot resistance etc. (Ayrilmis and Kaymakci, 2013). Apart from this, P. elongata is also known to show tolerance to a variety of biotic and abiotic stresses (Chaires et al., 2017). However not many studies are done on understanding its stress tolerance mechanism, which requires extraction of high-quality RNA.

High-quality RNA refers to RNA that is devoid of any genomic DNA, phenols, polysaccharides, secondary metabolites, etc. that interfere with molecular biology techniques (Ouyang et al., 2014). Genomic DNA contamination will affect the detection and quantification of gene expression analysis such as RT-PCR, qPCR, northern blotting and RNA sequencing (Añez-Lingerfelt et al., 2009). This is because the reaction cannot differentiate between cDNA (complementary DNA) and genomic DNA, which will lead to overestimation of gene expression present (Añez-Lingerfelt et al., 2009). Phenols in RNA can oxidize to form quinones that will bind to nucleic acids irreversibly (Ouyang et al., 2014). Polysaccharides and secondary metabolites can co-precipitate and degrade the RNA in the sample thereby affecting the yield, quality and downstream applications of RNA (Ouyang et al., 2014; Ghawana et al., 2011). Since P. elongata leaves are known to have a high content of alkaloids and proteins (Kirov et al., 2014), isolation of pure RNA from them poses a challenge. Methods using spin columns did not yield a good amount of RNA (data not shown) from P. elongata leaves, and this could be due to the fact that spin columns efficiency decreases in the presence of alkaloids (Ouyang et al., 2014). CTAB (cetyltrimethyl ammonium bromide) based methods are spin-column free but are time-consuming (Ouyang et al., 2014). Thus, we combined the Trizol (Invitrogen^TM) extraction method and spin-column based purification method in our protocol.

In our study we extracted high quantity RNA using a modified Trizol (Invitrogen^TM) method. The quality of RNA was improved by using RNA Clean and Concentrator Kit (ZYMO RESEARCH, USA) with modifications. The RNA yield was measured using Nanodrop spectrophotometer (Figure 1). The Nanodrop measurement peaks can also analyze the presence of phenols or polysaccharides in the sample. In our study, the peak indicated that the RNA was free from phenol or polysaccharide contamination (Figure 1). The integrity of the RNA was further affirmed by running an RNA gel (Formaldehyde free RNA gel kit, Amresco, USA). The gel revealed that the RNA extracted using our method was free from genomic DNA contamination. Clear 28S and 18S rRNA bands were observed (Figure 2). The RNA was then successfully used in downstream applications like RT-PCR and qPCR which amplified the ubiquitin gene (Figures 3 and 4). In RT-PCR, we used cDNA synthesized from our P. elongata RNA as the template. The cDNA was amplified using ubiquitin qPCR primers. The ubiquitin primers used in the study are as follows:
Forward Primer- 5’ GTC AGG AGG AAC ACC TTC TTT 3’
Reverse Primer- 5’ CCT TGA CTG GGA AGA CCA TTAC 3’

Thus bands of about 250 bp were observed as a result of successful amplification of ubiquitin (Figure 3). Our method of RNA isolation not only has high and pure yield but is also time-saving. The entire procedure takes about 2 h.