Biomaterial Database

Comparison of spermatozoal RNA extraction methods in goats. 2021

Bijayalaxmi Sahoo, and Praveen Kumar Guttula, and Mukesh Kumar Gupta

Gene Manipulation Laboratory, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha, 769 008, India.

RNA sequencing (RNAseq) has divulged newer role of spermatozoal RNA in male fertility. This study aimed to evaluate different sperm purification and RNA extraction methods for long-read RNA sequencing of poly(A) transcriptome in goat spermatozoa. Sperm samples were purified by swim-up separation using different purification medium. Spermatozoal RNA was extracted by seven different methods with additional supplementation of reducing agents in lysis buffer. poly(A) selected RNA was used for cDNA library preparation and long-read RNAseq in Nanopore sequencer. Sperm purification by 1 h swim-up resulted in higher recovery (89.20 ± 1.15%), motility (82.33 ± 1.53%), viability (88.10 ± 5.03%) and plasma membrane integrity (71.33 ± 4.51%) in sperm TALP (sp-TL) medium. A monophasic solution of GITC with phenol and DTT resulted in the highest yield of large sized RNAs (3.89 ± 0.46 ng/million cells) suitable for long-read RNAseq of poly(A) transcripts. RNAseq resulted in reads of length, ranging from 500bp to 2 Kb. A total of 123 transcripts were identified in goat spermatozoa by de novo assembly and included sperm-specific transcripts such as CATSPERG, PRM2, CYLC2, SPATA6, PLCZ1 etc. This study is the first report of long-read RNAseq of poly(A) transcriptome in goat spermatozoa.

UI	MeSH Term	Description	Entries
D008297	Male		Males
D005298	Fertility	The capacity to conceive or to induce conception. It may refer to either the male or female.	Fecundity,Below Replacement Fertility,Differential Fertility,Fecundability,Fertility Determinants,Fertility Incentives,Fertility Preferences,Fertility, Below Replacement,Marital Fertility,Natural Fertility,Subfecundity,World Fertility Survey,Determinant, Fertility,Determinants, Fertility,Fertility Determinant,Fertility Incentive,Fertility Preference,Fertility Survey, World,Fertility Surveys, World,Fertility, Differential,Fertility, Marital,Fertility, Natural,Preference, Fertility,Preferences, Fertility,Survey, World Fertility,Surveys, World Fertility,World Fertility Surveys
D006041	Goats	Any of numerous agile, hollow-horned RUMINANTS of the genus Capra, in the family Bovidae, closely related to the SHEEP.	Capra,Capras,Goat
D000081414	Nanopore Sequencing	A sequencing protocol that drives nucleic acids (DNA or RNA) in an electric field through NANOPORES allowing single molecule sequence analysis.	Nanopore Sequencings,Sequencing, Nanopore
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D012313	RNA	A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed)	RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D012333	RNA, Messenger	RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.	Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D013094	Spermatozoa	Mature male germ cells derived from SPERMATIDS. As spermatids move toward the lumen of the SEMINIFEROUS TUBULES, they undergo extensive structural changes including the loss of cytoplasm, condensation of CHROMATIN into the SPERM HEAD, formation of the ACROSOME cap, the SPERM MIDPIECE and the SPERM TAIL that provides motility.	Sperm,Spermatozoon,X-Bearing Sperm,X-Chromosome-Bearing Sperm,Y-Bearing Sperm,Y-Chromosome-Bearing Sperm,Sperm, X-Bearing,Sperm, X-Chromosome-Bearing,Sperm, Y-Bearing,Sperm, Y-Chromosome-Bearing,Sperms, X-Bearing,Sperms, X-Chromosome-Bearing,Sperms, Y-Bearing,Sperms, Y-Chromosome-Bearing,X Bearing Sperm,X Chromosome Bearing Sperm,X-Bearing Sperms,X-Chromosome-Bearing Sperms,Y Bearing Sperm,Y Chromosome Bearing Sperm,Y-Bearing Sperms,Y-Chromosome-Bearing Sperms
D015723	Gene Library	A large collection of DNA fragments cloned (CLONING, MOLECULAR) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences.	DNA Library,cDNA Library,DNA Libraries,Gene Libraries,Libraries, DNA,Libraries, Gene,Libraries, cDNA,Library, DNA,Library, Gene,Library, cDNA,cDNA Libraries
D017423	Sequence Analysis, RNA	A multistage process that includes cloning, physical mapping, subcloning, sequencing, and information analysis of an RNA SEQUENCE.	RNA Sequence Analysis,Sequence Determination, RNA,Analysis, RNA Sequence,Determination, RNA Sequence,Determinations, RNA Sequence,RNA Sequence Determination,RNA Sequence Determinations,RNA Sequencing,Sequence Determinations, RNA,Analyses, RNA Sequence,RNA Sequence Analyses,Sequence Analyses, RNA,Sequencing, RNA