As a medium for receiving, transmitting and responding to numerous biological signals, blood is involved in the metabolism, monitoring, maintenance of metabolism and immune function of almost all cells, tissues and organs. It is an easy-to-obtain clinical sample and it plays a vital role in the research of blood diseases, immunity, cancer, endocrine and other aspects. The rRNA content in total RNA of peripheral blood is more than 90%, and Globin mRNA accounts for about 70% of mRNA. These RNA genes can provide less effective information. The direct use of blood for sequencing not only produces a lot of worthless data, but also affects the detection of low abundance mRNA. Therefore, the effective removal of rRNA and Globin mRNA is the first important step for RNA library construction.
Ribo-off Globin & rRNA Depletion Kit (Human/Mouse/Rat) (N408) is for Blood Total RNA samples, through the steps of the hybridization of Globin mRNA and rRNA with probe, RNase H depletion, DNase I depletion and other steps, finally they are removed from Total RNA, and other mRNAs and non-coding RNAs are retained, which can be used for the analysis of non-coding RNAs such as LncRNA. Applicable sample types are abundant, compatible with low starting amount (0.01-1 μg) and low-quality samples. It can efficiently remove rRNA and Globin mRNA, and significantly improve the effective data information in sequencing data and the detection rate of low-abundance samples.
Principle of the Experiment
(1) The starting amount should be as low as 10 ng
Set different initial input levels, use N408 to remove rRNA and Globin mRNA, and build and sequence the removed products to obtain high-quality Clean Reads. Compared to the number of Reads without the removal of rRNA genes and Globin mRNA genes and after using other companies’ products to remove rRNA genes and Globin mRNA genes, the results show that with different starting amounts (0.01-1μg), N408 can efficiently remove the target RNA.
Figure 2. Removal effect of Globin mRNA and rRNA with different starting amounts
(2)Removing rRNA and Globin mRNA from different types of samples and using the comparison software (BWA) to compare the sequencing data to the rRNA Sequencing database of their respective species, the results show that N408 can efficiently remove target RNA for samples of different species.
Figure 3. Globin mRNA and rRNA removal in different species
(2)Efficient removal of low-quality samples
Removing rRNA and Globin mRNA from samples with different RIN values, and performing library construction and sequencing on the removed products to obtain the number of Reads before and after using our company to remove the Globin mRNA genes and rRNA genes, and using other companies’ products to remove the Globin mRNA genes and rRNA genes, the results show that samples with different RIN values can be removed efficiently by N408 Target RNA.
Figure 4. Globin mRNA and rRNA removal effect of samples with different RIN values
Remove rRNA and Globin mRNA from samples with different starting amounts (0.01-1 μg) and different species, and use the new product - VAHTS® Universal V8 RNA-seq Library Prep Kit for Illumina (#NR605) for transcriptome library construction. Based on the comparison result of HISAT2, we use stringtie to complete the quantitative analysis of gene expression and count the number of gene detections. The results show that different starting amounts (0.01-1 μg) and samples of different species can guarantee a rich number of gene detections.
Different Starting Amount (Human)
Different species (100 ng)
Figure 5. The number of genes detected in samples of different starting amounts and different species