Alveolar Bone RNA Extraction and Gene Expression in Health and Diabetes

There is a gap in the literature regarding RNA extraction and human alveolar bone gene expression in type 2 diabetes.The overall objective of this novel study is to investigate the gene expression from alveolar bone in health and diabetes.There are two broad objectives;1To improve RNA extraction protocol from human alveolar bone and improve RIN (RNA integrity values) 2 Analyze gene expression profiles in health and diabetes RNA sequencing.Methods-Healthy and Type 2 diabetic subjects undergoing periodontal surgeries were screened and consented.The subjects were undergoing procedures like osseous surgery,tori removal,crown lengthening,extraction and alveoplasty were selected. A total of 43 subjects were recruited until now for the study (25 healthy and 18 diabetes).At the surgery appointment, alveolar bone was harvested and immediately placed in a vial containing 500ul of Trizol and immediately frozen in liquid nitrogen.Multiple vials were obtained if there was more bone available.Samples were processed within two months of collection.During the RNA extraction stage, bone samples were homogenized with a bone crusher in liquid nitrogen to uniformly break larger pieces of bone and then were processed in a bullet blender with metal beads in a cold room.The homogenized sample was extracted and processed by Qiagen mini columns.RNA was then analyzed on nano-drop spectrophotometer for quality and quantity and a bio-analyzer to obtain RNA integrity number(RIN).Samples that had greater than 3.5 RIN were then processed by a rRNA depletion method to further purify the samples and were submitted for RNA sequencing for differential gene expression.Results-Extracting stable and high quality RNA from human bone samples is technique and patient dependent.Our data showed that maintaining low or below freezing temperatures of the bone samples are key to good RIN values that may influence techniques like RT-PCR and RNA sequencing. Until now 25 healthy and 18 diabetics subjects were recruited.Our results show that a total of 10 healthy and 8 diabetic samples had RIN Values of 3.5 or more and were submitted for RNA sequencing for differential gene expression.The mean age, HbA1c% and RIN values of healthy vs diabetic subjects is;age (55.3 ± 17.5 vs 63.9 ± 8.7 years), HbA1c% ( 5.6 ± 0.29 vs 7.3 ± 2.4) and RIN ( 4.94 ± 1.41 vs 4.43 ± 1.54) respectively. Sequencing analysis shows that overall gene expression was downregulated in diabetes samples. Conclusions-Understanding alveolar gene expression in diabetes is very important its molecular and clinical effects on periodontal regeneration or dental implants are relatively unknown.Our results here showed that our protocol for RNA extraction from human bone samples results in improved RIN values. Higher RIN values (> 6) are preferred for sensitive gene expression techniques like RT-PCR and RNA sequencing, however methods like rRNA depletion techniques are available to purify and amplify the RNA samples. Data from RNA sequencing shows that overall gene expression and specific pathways for inflammation and bone regulation were down regulated in diabetic subjects compared to healthy controls. Palabras clave: bone, diabetes, genes