Introduction

RNA sequencing (RNA-seq) has quickly become the standard method for transcriptomics (Wang et al., 2009; Metzker, 2010) and has been further developed into a number of modified protocols that allow detection from plants (van Dijk et al., 2014). The power of RNA-seq to reveal cell population heterogeneity in transcriptome-wide fashion has made it the focus of intense recent research activity aimed at its further development and on analysis techniques (Nagalakshmi et al., 2008; Parchman et al., 2010). It has made it possible that the overall analysis of genomes, transcriptomes and interactomes become less expensive, routine and widespread, which has greatly contributed to problem solving in various biological and biomedical research (Shendure and Ji, 2008; Trapnell et al., 2010).

Sample processing for RNA-seq is largely based on traditional molecular biological protocols that were developed decades ago for common assays such as RT-PCR, Northern blotting or microarrays. The typical workflow of an RNA-seq assay involves the extraction (and often further purification) of mRNA from plants, the preparation of a sequencing library including poly-(A)⁺RNA isolation, fragmentation, adaptor ligation, reverse transcription, and PCR amplification, next-generation sequencing, and computational processing and analysis of the resulting data (Metzker, 2010; Marine et al., 2011; Hebenstreit, 2012). The selection of poly-(A)+ RNA is usually performed in order to suppress the “loss” of sequencing reads to structural RNAs such as rRNA and tRNA, which represent the bulk of cellular RNA (Marzluff, 2008). The fragmentation step is carried out in order to produce many short mRNA fragments that represent the original transcript. An ideal fragmentation protocol should be inexpensive to perform, have a simple workflow, and be amenable to automation (Sigman et al., 1993; Kelly et al., 2002). Fragmentation is typically achieved either by mechanical force through nebulization or sonication, or by enzymatic digestion (Erwin and van Dijka, 2014). Depending on the exact protocol, the fragmentation serves to optimize the length for the sequencing machine.

1 Results

1.1 The extraction of RNA

Library construction is the preparation of the nucleic acid target into a form that is compatible with the sequencing system to be used (Head et al., 2014) (Figure 1A). Sample processing is largely based on traditional molecular biological protocols that were developed decades ago for common assays such as RT-PCR, Northern blotting or microarrays (Head et al., 2014). The basic workflow of mRNA islolation and library preparation is shown in Figure 1B and Figure 1C, respectively.

Figure 1 The basic workflow of library preparation for NGS analysis Note: A: The core steps  in preparing RNA or DNA for NGS analysis (Head et al., 2014); B: Schematic overview of the poly(A) RNA selection from tissue sample using Dynabeads Oligo(dT)25; C: The Outline of the protocol for isolating mRNA (Dynabeads ® mRNA DIRECT ™ Kitprotocol, Catalog numbers 61011)

For all of the RNA samples from our experiments, they all had distinct 28S, 18S and 5S bands without degradation (Figure 2A; Figure 2B). The A260/280 absorbance ratio was between 2.0 to 2.1 and the RIN value was above 8.30. So the RNA extracted using this method could be used for further analyses as demonstrated by transcriptome construction. Thus this method is an efficient and reproducible procedure for the isolation of RNA from 84K Poplar.

Figure 2 Quality and quantity assessment using Agilent 2100 Bioanalyzer and agarose gel electrophoresis Note: A: Test results of agilent 2100 Bioanalyzer; B: Test results of agarose gel electrophoresis of the libraries

1.2 Quality inspection of results of the transcriptome libraries

The major goal of RNA-seq is to accurately reveal the relative expression levels of the original mRNA (Hebenstreit, 2011; Cloonan, 2008), because rRNA reads are not informative for most RNA-Seq experiments (Yi, 2011).Therefore it is best to reduce the levels of rRNA. The size of the target DNA fragments in the final library is a key parameter for RNA-Seq library construction. The result of the Agilent 2100 analysis indicates that the central value is bteween 400~600 bp, which could meet the requirement of on-machine (Figure 3).

Figure 3 Test results of Agilent 2100 Bioanalyzer after the purification option of mRNA for constructing transcriptome libraries Note: A: Electropherogram summary of peak table for ladder; B: The results of Agilent 2100 Bioanalyzer

2 Discussion

A major bottleneck in RNA sequencing is library construction (Steven, 2015), Analyzing RNA is more challenging than analyzing DNA, because double-stranded DNA is more stable than single-stranded RNA, Moreover, deoxyribonucleases (DNases) are readily denatured and inhibited compared to the highly stable ribonucleases (RNAse) (Azevedo, 2003; Wang, 2007; Gudenschwager, 2012).

The extraction of mRNA from plant is difficult due to various limitations e.g. the shorter half life time of mRNA compared to rRNA ranging between 30 s and 20 min (Ehretsmann, 1992; Sharma, 2012),The contents of mRNA  is less than 5% in total RNA extracted from plant, especially in the case of RNA-seq, which is technically more challenging than DNA-seq (Turashviliand Yang, 2012) and making it difficult to apply to woody plants. So, in our experiment, four methods were used to assess the relative effectiveness for the isolation of good quality RNA.

In our experiment all samples were analyzed on the Agilent 2100 bioanalyzer. The Agilent 2100 bioanalyzer, a bio-analytical device based on a combination of microfluidics, microcapillary electrophoresis, and fluorescence detection, provides a platform to record the size distribution of molecules. The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. High gene expression (intact RNA) shown by high RIN value while low gene expression (degraded RNA) is shown by a low RIN value.

The size of the target DNA fragments in the final library is a key parameter for RNA-Seq library construction. Fragmentation is essential factor and most library preparation protocols use fragmentation for the detection of libraries, since it influences the insert size and leads to different insert size by the detection of library.

The results showed that the degree of purification of mRNA obviously influenced the fragmentation and size selector which were detected by Agilent 2100 Bioanalyzer and also showed that when the transcriptome libraries were constructed from leaves of Populus, the option of 2.5 M LiCl binding buffer and 0.1 M LiCl elution buffer combined with 1% of LiDS could thoroughly remove the rRNA and other impurities to obtain complete, high-purity mRNA molecules. The insights into molecular reactions that our framework allows can be further exploited to improve RNA-seq protocols, as we demonstrated experimentally.

3 Materials and Methods

3.1 Plant materials and Chemicals and reagents

The study was carried out on the sterile plantlets of Poplar 84K (Populus alba X Populus glandulosa) which were acquired from State Key Laboratory of Tree Genetics and Breeding in Northeast Forestry University in Harbin, Heilongjiang. The following chemicals and reagents were used in specific proportion for the isolation and purification of mRNA. Dynabeads Oligo(dT)₂₅ (Thermo Corporation), Magnetic beads (Thermo Corporation), magnetic frame , LiCl (Sigma Corporation), Lithium Dodecyl Sulfa(LiDS) (Beijing Dingguo Biotechnology Co., Ltd), RNA Seq Library Preparation Kit (Nanjing Keweisi Technology Co., Ltd), RNaseOU^TMRecombinant Ribonuclease inhibitor (Invitrogen), EDTA disodium salt solution (0.5 M; Sigma), CTAB, NaCl, Tris-HCl (PH 8.0), Chloroform, Alcohol, DTT, Sodium azide, and PVP40.

3.2 RNA analysis

The concentration and purification of RNA in RNase-free water was evaluated at 230, 260 and 280 nm using NanoDrop spectrophotometry (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀). The RNA integrity was assessed by the sharpness of ribosomal RNA bands visualized on a denaturing 1.2% agarose gel. RNA integrity was further evaluated in an Agilent 2100 BioAnalyzer (Agilent Technologies), which uses automated microfluidics capillary electrophoresis and fluorescence to evaluate RNA integrity.

3.3 Isolation of mRNA

20 uL of eluted mRNA from the first purification was transferred to a new RNase free tube and placed on ice and the beads were not discarded. The beads were washed twice with the appropriate volume of Washing Buffer B (1~1.5 mL) at room temperature and the magnet was used to separate the beads from the solution. The eluted mRNA was diluted with 4 times its volume of Lysis Binding Buffer (e.g., if the mRNA was eluted in 20 uL, 80 uL of Lysis or Binding Buffer was added). Washing Buffer B was removed from the beads by placing the tube on the magnet and adding the diluted mRNA. The mixture was then incubated at room temperature for 3~5 minutes. During incubation, there was continuous mixing (rotating or roller mixer) for 3~5 min at room temperature to allow the polyA tail of the mRNA to hybridize with the oligo (dT) ₂₅ on the beads. Note: Incubation time was increased for up to 10 minutes if the solutions were noticeably viscous. The vial was later placed on the magnet for 2 minutes and the supernatant removed. The beads/mRNA complex was washed two times with the appropriate volume of Washing Buffer A at room temperature. In each washing step, the magnet was used to separate the beads from the solution. The beads/mRNA complex was washed once more with the appropriate volume of Washing Buffer B at room temperature. To elute mRNA from the beads, the Washing Buffer B was removed and 10~20 μL 10 mM Tris-HCl was added and then incubated at 75°C to 80°C for 2 min. The tube was then placed on the magnet and quickly transferred the supernatant containing the mRNA to a new RNase-free tube. The buffers and solutions of the option were shown Wang et al. (2018) (doi:gmx130).

3.4 The construction of Transcriptome Library

The Library preparation was performed following the RNA Seq Library Preparation Kit for Transcriptome Discovery Manual with the following steps: (1) fragmentation of mRNA, (2) Ligation of 5’ adaptor, (3) Reverse transcription, (4) PCR amplification, and (5) Size selector (Yin and Wang, 2013).

4 Conclusion

The degree of mRNA purification affects the process of fragmentation, which further affects the final insert size of the library, the experiments show that under the second circumstance, the combination between Buffer (2.5 M LiCl, 2 mM EDTA) and Lysis/Binding Buffer (500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM DTT) together with the combination between Washing Buffer A (0.25 M LiCl, 1 mM EDTA, 0.5% LiDS) and Washing Buffer B (0.15 M LiCl, 1 mM EDTA,10 mM Tris-HCl), respectively. The result of the Agilent 2100 analysis indicates that the central value is between 400-600 bp, which could meet the requirement of on-machine.

Authors' contributions

L.N.W. conceived, designed and performed the experiments. Y.F.L. analyzed the data and supervised the project. L.F.W. offered technical assistance and statistical analyses. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by the Fundamental Research Funds for the Central Universities (2572016BA02) and Harbin applied technology research and development project (2016RAQXJ065),we greatly appreciate Professor Wang Baichen and Professor Wang Yucheng’ s assistance in these experiments.

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